Nucleic acid molecules encoding osteoclastogenesis inhibitory factor proteins

ABSTRACT

A protein which inhibits osteoclast differentiation and/or maturation and a method for producing the protein. The protein is produced by human embryonic lung fibroblasts and has a molecular weight of about 60 kD and about 120 kD under non-reducing conditions and about 60 kD under reducing conditions on SDS-polyacrylamide gel electrophoresis. The protein can be isolated and purified from the culture medium of fibroblasts. Furthermore, the protein can be produced by gene engineering. The present invention includes cDNA for producing the protein by gene engineering, antibodies having specific affinity for the protein or a method for determining protein concentration using these antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 10/232,858, filedSep. 3, 2002, now U.S. Pat. No. 6,855,808 which is a continuation ofU.S. application Ser. No. 08/915,004, filed Aug. 20, 1997, now U.S. Pat.No. 7,125,686 which is a continuation-in-part of internationalapplication PCT/JP96/00374, filed Feb. 20, 1996, which claims priorityto Japanese applications JP/054977, filed Feb. 20, 1995 and JP/207508,filed Jul. 21, 1995.

INCORPORATION OF SEQUENCE LISTING

Herein incorporated by reference is the Sequence Listing, which has beensubmitted on paper and diskette as a file named “SubSeq16991018.txt”which is 136,653 bytes in size (measured in MS-DOS), and which wascreated on Aug. 27, 2004.

FIELD OF THE INVENTION

This invention relates to a novel protein, osteoclastogenesis inhibitoryfactor (OCIF), and methods for producing the protein.

BACKGROUND OF THE INVENTION

Human bones are always remodelling by the repeated process of resorptionand reconstitution. Osteoblasts and osteoclasts are considered to be thecells mainly responsible for bone formation and bone resorption,respectively. A typical example of a disease caused by abnormal bonemetabolism is osteoporosis. Osteoporosis is known to develop when boneresorption by osteoclasts exceeds bone formation by osteoblasts, but themechanism of osteoporosis has not yet been completely elucidated.Osteoporosis causes bone pain and makes bones fragile, leading tofracture, particularly in elderly patients. Osteoporosis has thereforebecome a social issue with the increasing number of elderly people inthe population. Therefore, effective drugs for the treatment of thedisease are expected to be developed. Bone mass reduction caused byabnormal bone metabolism is thought to be prevented by inhibiting boneresorption, improving bone formation, or improving the balance of bonemetabolism.

Bone formation is promoted by stimulating growth, differentiation, oractivation of osteoblasts. Many cytokines reportedly stimulate growth ordifferentiation of osteoblasts, i.e. fibroblast growth factor (FGF)(Rodan S. B. et al., Endocrinology vol. 121, p 1917, 1987), insulin-likegrowth factor-I (IGF-I) (Hock J. M. et al., Endocrinology vol. 122, p254, 1988), insulin-like growth factor-II (IGF-II) (McCarthy T. et al.,Endocrinology vol. 124, p 301, 1989), Activin A (Centrella M. et al.,Mol. Cell Biol. vol. 11, p 250, 1991), Vasculotropin (Varonique M. etal., Biochem. Biophys. Res. Commun. vol. 199, p 380, 1994), and bonemorphogenetic protein (BMP) (Yamaguchi, A. et al., J. Cell Biol. vol.113, p 682, 1991, Sampath T. K. et al., J. Biol. Chem. vol. 267, p20532, 1992, and Knutsen R. et al., Biochem. Biophys. Res. Commun. vol.194, p 1352, 1993).

On the other hand, cytokines which inhibit differentiation and/ormaturation of osteoclasts have also been intensively studied.Transforming growth factor-β (Chenu C. et al., Proc. Natl. Acad. Sci.USA, vol. 85, p 5683, 1988) and interleukin-4 (Kasano K. et al.,Bone-Miner., vol. 21, p 179, 1993) inhibit the differentiation ofosteoclasts. Calcitonin (Bone-Miner., vol. 17, p 347, 1992), macrophagecolony-stimulating factor (Hattersley G. et al., J. Cell. Physiol. vol.137, p 199, 1988), interleukin-4 (Watanabe, K. et al., Biochem. Biophys.Res. Commun. vol. 172, p 1035, 1990), and interferon-γ (Gowen M. et al.,J. Bone-Miner. Res., vol. 1, p 469, 1986) inhibit bone resorption byosteoclasts.

These cytokines are expected to be effective drugs for improving bonemass reduction by stimulating bone formation and/or by inhibiting boneresorption. Cytokines such as insulin like growth factor-I and bonemorphogenetic proteins have been investigated in clinical trials fortheir effectiveness for treating patients with bone diseases. Calcitoninis already used for osteoporosis and to diminish pain in osteoporosispatients.

Examples of drugs now clinically utilized for the treatment of bonediseases and for shortening the treatment period are dihydroxyvitamineD₃, vitamin K₂, calcitonin and its derivatives, hormones such asestradiol, ipriflavon, and calcium preparations. However, these drugs donot provide satisfactory therapeutic effects, and novel drug substancesare expected to be developed. Since bone metabolism is manifest in thebalance between bone resorption and bone formation, cytokines whichinhibit osteoclast differentiation and/or maturation are expected to bedeveloped as drugs for the treatment of bone diseases such asosteoporosis.

SUMMARY OF THE INVENTION

The purpose of this invention is to offer both a novel factor, termedosteoclastogenesis inhibitory factor (OCIF), and a procedure to producethe factor efficiently.

The inventors have intensively searched for osteoclastogenesisinhibitory factors in human embryonic fibroblast IMR-90 (ATCC CCL186)conditioned medium and have found a novel osteoclastogenesis inhibitoryfactor (OCIF) which inhibits differentiation and/or maturation ofosteoclasts.

The inventors have established a method for accumulating the protein toa high concentration by culturing IMR-90 cells on alumina ceramicpieces, which function as cell adherence matrices.

The inventors have also established an efficient method for isolatingthe protein, OCIF, from the IMR-90 conditioned medium using thefollowing sequential column chromatography: ion-exchange, heparinaffinity, cibacron-blue affinity, and reverse phase.

After determining the amino acid sequence of the purified natural OCIF,a cDNA encoding this protein was successfully cloned. A procedure forproducing this protein was also established. The invention concerns aprotein which is produced by human lung fibroblast cells, has amolecular weight by SDS-PAGE of 60 kD under reducing conditions andmolecular weights of 60 kD and 120 kD under non-reducing conditions, andhas affinity for both cation-exchange resins and heparin. The protein'sability to inhibit the differentiation and maturation of osteoclasts isreduced when treated for 10 minutes at 70° C. or for 30 minutes at 56°C., and its ability to inhibit differentiation and maturation ofosteoclasts is lost when treated for 10 minutes at 90° C. The amino acidsequence of the OCIF protein of the present invention is clearlydifferent from any other factors known to inhibit the formation ofosteoclasts.

The invention includes a method for purifying OCIF protein, comprising:(1) culturing human fibroblasts, (2) applying the conditioned medium toa heparin column to obtain the adsorbed fraction, (3) purifying the OCIFprotein using a cation-exchange column, (4) purifying the OCIF proteinusing a heparin affinity column, (5) purifying the OCIF protein using aCibacron blue affinity column, and (6) isolating the OCIF protein usingreverse-phase column chromatography. Cibacron blue F3GA dye may becoupled to a carrier made of synthetic hydrophilic polymers, forexample, to form columns conventionally called “blue columns”.

The invention includes a method for producing OCIF protein in highconcentration by culturing human fibroblasts using alumina ceramicpieces as the cell-adherence matrices.

Moreover, the inventors determined the amino acid sequences of peptidesderived from OCIF, designed the oligonucleotide primers based on theseamino acid sequences, and obtained cDNA fragments encoding OCIF from acDNA library of IMR-90 human fetal lung fibroblast cells. The fulllength OCIF cDNA encoding the OCIF protein is cloned from a cDNA libraryusing an OCIF DNA fragment as a probe. The OCIF cDNA containing theentire coding region is inserted into an expression vector. RecombinantOCIF can be produced by expressing the OCIF cDNA, containing the entirecoding region, in mammalian cells or bacteria.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the elution pattern of crude OCIF protein(HILOAD™-Q/FF pass-through fraction; sample 3) from a HILOAD™-S/HPcolumn.

FIG. 2 shows the elution pattern of crude OCIF protein (heparin-5PWfraction; sample 5) from a blue-5PW column.

FIG. 3 shows the elution pattern of OCIF protein (blue-5PW fraction 49to 50) from a reverse-phase column.

FIGS. 4A and 4B show the SDS-PAGE results of isolated OCIF proteinsunder reducing or non-reducing conditions. Description of the lanes:

-   -   lane 1, 4: molecular weight marker proteins;    -   lane 2, 5: OCIF protein of peak 6 in FIG. 3;    -   lane 3, 6: OCIF protein of peak 7 in FIG. 3.

FIG. 5 shows the elution pattern of peptides (peak 7) obtained by thedigestion of pyridyl ethylated OCIF protein digested withlysylendopeptidase, on a reverse-phase column.

FIG. 6 shows the SDS-PAGE results of isolated natural (n) OCIF proteinand recombinant (r) OCIF proteins under non-reducing conditions. rOCIF(E) and rOCIF (C) proteins were produced by 293/EBNA cells and by CHOcells, respectively. Description of the lanes:

-   -   lane 1: molecular weight marker proteins;    -   lane 2: a monomer type nOCIF protein;    -   lane 3: a dimer type nOCIF protein;    -   lane 4: a monomer type rOCIF (E) protein;    -   lane 5: a dimer type rOCIF (E) protein;    -   lane 6: a monomer type rOCIF (C) protein;    -   lane 7: a dimer type rOCIF (C) protein.

FIG. 7 shows the SDS-PAGE results of isolated natural (n) OCIF proteinsand recombinant (r) OCIF proteins under reducing conditions. rOCIF (E)and rOCIF (C) were produced by 293/EBNA cells and by CHO cells,respectively. Description of the lanes:

-   -   lane 8: molecular weight marker proteins;    -   lane 9: a monomer type nOCIF protein;    -   lane 10: a dimer type nOCIF protein;    -   lane 11: a monomer type rOCIF (E) protein;    -   lane 12: a dimer type rOCIF (E) protein;    -   lane 13: a monomer type rOCIF (C) protein;    -   lane 14: a dimer type rOCIF (C) protein.

FIG. 8 shows the SDS-PAGE results of isolated natural (n) OCIF proteinsand recombinant (r) OCIF proteins from which N-linked sugar chains wereremoved under reducing conditions. rOCIF (E) and rOCIF (C) are rOCIFproteins produced by 293/EBNA cells and by CHO cells, respectively.Description of the lanes:

-   -   lane 15: molecular weight marker proteins;    -   lane 16: a monomer type nOCIF protein;    -   lane 17: a dimer type nOCIF protein;    -   lane 18: a monomer type rOCIF (E) protein;    -   lane 19: a dimer type rOCIF (E) protein;    -   lane 20: a monomer type rOCIF (C) protein;    -   lane 21: a dimer type rOCIF (C) protein.

FIG. 9 shows a comparison of OCIF (SEQ ID NO: 5) and OCIF2 (SEQ ID NO:9) amino acid sequences.

FIG. 10 shows a comparison of OCIF (SEQ ID NO: 5) and OCIF3 (SEQ ID NO:11) amino acid sequences.

FIG. 11 shows a comparison of OCIF (SEQ ID NO: 5) and OCIF4 (SEQ ID NO:13) amino acid sequences.

FIG. 12 shows a comparison of OCIF (SEQ ID NO: 5) and OCIF5 (SEQ ID NO:15) amino acid sequences.

FIG. 13 shows a standard curve determining OCIF protein concentration byan EIA employing anti-OCIF polyclonal antibodies.

FIG. 14 shows a standard curve determining OCIF protein concentration byan EIA employing anti-OCIF monoclonal antibodies.

FIG. 15 shows the effect of rOCIF protein on model rats withosteoporosis.

DETAILED DESCRIPTION OF THE INVENTION

The OCIF protein of the present invention can be isolated from humanfibroblast conditioned medium with high yield. The procedure to isolateOCIF is based on ordinary techniques for purifying proteins frombiomaterials, in accordance with the physical and chemical properties ofOCIF protein. For example, concentrating procedures include ordinarybiochemical techniques such as ultrafiltration, lyophilization, anddialysis. Purifying procedures include combinations of severalchromatographic techniques for purifying proteins such as ion-exchangecolumn chromatography, affinity column chromatography, gel filtrationcolumn chromatography, hydrophobic column chromatography, reverse phasecolumn chromatography, and preparative gel electrophoresis. The humanfibroblasts used for the production of OCIF protein are preferablyIMR-90 cells. A method for producing IMR-90 conditioned medium ispreferably a process comprising adhering human embryonic fibroblastIMR-90 cells to alumina ceramic pieces in roller-bottles in DMEM mediumsupplemented with 5% newborn calf serum, and cultivating the cells inroller-bottles for 7 to 10 days by stand cultivation. CHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) ispreferably added to the buffer as a detergent in the proteinpurification procedure.

The OCIF protein of the instant invention can be obtained initially as abasic heparin binding OCIF fraction by applying the culture medium to aheparin column (Heparin-SEPHAROSE™ CL-6B, Pharmacia), eluting with 10 mMTris-HCl buffer, pH 7.5, containing 2 M NaCl, and applying the OCIFfraction to a Q • anion-exchange column (HILOAD™-Q/FF, Pharmacia), andcollecting the non-adsorbed fraction. OCIF protein can be purified bysubjecting the obtained OCIF fraction to purification on an S •cation-exchange column (HILOAD™-S/FF, Pharmacia), a heparin column(Heparin-5PW, TOSOH), a Cibacron Blue column (Blue-5PW, TOSOH), and areverse-phase column (BU-300 C4, Perkin Elmer).

The present invention relates to a method of cloning cDNA encoding theOCIF protein based on the amino acid sequence of natural OCIF and amethod for obtaining recombinant OCIF protein. The OCIF protein ispurified according to the method described in the present invention andis treated with endopeptidase (for example, lysylendopeptidase). Theamino acid sequences of the peptides produced by the digestion aredetermined and the mixture of oligonucleotides that can encode eachinternal amino acid sequence is synthesized. The OCIF cDNA fragment isobtained by PCR (preferably RT-PCR, reverse transcriptase PCR) using theoligonucleotide mixtures described above as primers. The full lengthOCIF cDNA encoding the OCIF protein is cloned from a cDNA library usingan OCIF DNA fragment as a probe. The OCIF cDNA containing the entirecoding region is inserted into an expression vector. Recombinant OCIFcan be produced by expressing the OCIF cDNA, containing the entirecoding region, in mammalian cells or bacteria.

The present invention relates to the novel proteins OCIF2, OCIF3, OCIF4,and OCIF5 that are variants of OCIF and have the activity describedabove. These OCIF variants are obtained from the cDNA libraryconstructed with IMR-90 poly(A)+RNA using the OCIF cDNA fragment as ahybridization probe. Each of the OCIF variant cDNAs containing theentire coding region is inserted into an expression vector. Eachrecombinant OCIF variant protein can be produced by expressing each ofthe OCIF variant cDNAs, containing the entire coding region, inconventional hosts. Each recombinant OCIF variant protein can bepurified according to the method described in this invention. Eachrecombinant OCIF variant protein has the ability to inhibitosteoclastogenesis.

The present invention further includes OCIF mutants. They aresubstitution mutants comprising the replacement of one cysteine residue,possibly involved in dimer formation, with a serine residue or variousdeletion mutants of OCIF. Substitutions or deletions are introduced intothe OCIF cDNA using polymerase chain reaction (PCR) or restrictionenzyme digestion. Each of these mutated OCIF cDNAs is inserted into avector having an appropriate promoter for gene expression. The resultantexpression vector for each of the OCIF mutants is introduced intoeukaryotic cells such as mammalian cells. Each of OCIF mutants can beobtained and purified from the conditioned media of the transfectedcells.

The present invention provides polyclonal antibodies and a method toquantitatively determine OCIF concentration using these polyclonalantibodies.

Natural OCIF obtained from IMR-90 conditioned medium, recombinant OCIFproduced by such hosts as microorganisms and eukaryotes using OCIF cDNA,synthetic peptides based on the amino acid sequence of OCIF, or peptidesobtained from OCIF by partial digestion can be used as antigens.Anti-OCIF polyclonal antibodies are obtained by immunizing appropriatemammals with the antigens, in combination with adjuvants if necessary,and purifying the antibodies from the serum by ordinary purificationmethods. Anti-OCIF polyclonal antibodies which are labelled withradioisotopes or enzymes can be used in radio-immunoassay (RIA) systemsor enzyme-immunoassay (EIA) systems. Using these assay systems, theconcentration of OCIF in biological materials such as blood, ascites andcell-culture medium can be easily determined.

The present invention provides novel monoclonal antibodies and a methodfor quantitatively determining OCIF concentration using these monoclonalantibodies.

Anti-OCIF monoclonal antibodies can be produced by conventional methodsusing OCIF as an antigen. Native OCIF obtained from the culture mediumof IMR-90 cells and recombinant OCIF produced by such hosts asmicroorganisms and eukaryotes transfected with OCIF cDNA can be used asantigens. Alternatively, synthetic peptides based on the amino acidsequence of OCIF and peptides obtained from OCIF by partial digestioncan be also used as antigens. Immunized lymphocytes obtained byimmunizing mammals such as mice or rats with the antigen or by an invitro immunization method were fused with mammalian myeloma cells toobtain hybridomas. The hybridoma clones secreting antibodies whichrecognize OCIF were selected and cultured to obtain the desiredantibodies. For immunizations, OCIF is suitably diluted with a salinesolution (0.15 M NaCl), and is intravenously or intraperitoneallyadministered with an adjuvant to animals 2-5 times every 2-20 days. Theimmunized animal was killed three days after the final immunization, thespleen was removed and the splenocytes were used as immunized Blymphocytes.

Mouse myeloma cell lines useful for cell fusion with immunized Blymphocytes include, for example, p3/x63-Ag8, p3-U1, NS-1, MPC-11,SP-2/0, FO, p3x63 Ag8.653, and S194 cells. The rat cell line R-210 mayalso be used. Alternatively human B lymphocytes immunized by an in vitroimmunization method are fused with human myeloma cells or EB virustransformed human B lymphocytes to produce human type antibodies.

Cell fusion of immunized B lymphocytes and myeloma cells is carried outprincipally by conventional methods. For example, the method of KoehlerG. et al. (Nature 256, 495-497, 1975) is generally used. Alternatively,an electric pulse method can be used. The immunized B lymphocytes andtransformed B cells are mixed at conventional ratios and a cell culturemedium without FBS containing polyethylene glycol is generally used tofuse the cells. The fusions products are cultured in HAT selectionmedium containing FBS to select hybridomas.

An EIA, plaque assay, Ouchterlony, or agglutination assay can be used toscreen for hybridomas producing anti-OCIF antibodies. EIA is a simpleassay which is easy to perform with sufficient accuracy and is thereforegenerally used. The desired antibody can be selected easily andaccurately using EIA and purified OCIF. Hybridomas obtained thereby canbe cultured by conventional methods of cell culture and frozen for stockif necessary. The antibody can be produced by culturing hybridoma cellsusing ordinary cell culture methods or by transplanting hybridoma cellsintraperitoneally into live animals. The antibody can be purified byordinary purification methods such as salt precipitation, gelfiltration, and affinity chromatography. The antibody obtainedspecifically reacts with OCIF and can be used to determine OCIFconcentration and to purify OCIF protein. The antibodies of the presentinvention recognize epitopes of OCIF and have high affinity for OCIF.Therefore, they can be used for the construction of EIA. This assaysystem is useful for determining the concentration of OCIF in biologicalmaterials such as blood and ascites.

The present invention provides agents containing OCIF as an effectiveingredient, that are useful for treating bone diseases. Rats weresubjected to denervation of the left forelimb. Test compounds wereadministered daily after surgery for 14 days. After 2 weeks oftreatment, the animals were sacrificed and their forelimbs weredissected. Thereafter bones were tested for mechanical strength by thethree point bending method. OCIF improved the mechanical strength ofbone in a dose dependent manner.

The OCIF protein of the invention is useful as a pharmaceuticalingredient for treating or improving decreased bone mass in bonediseases such as osteoporosis, rheumatism, osteoarthritis, and abnormalbone metabolism in multiple myeloma. OCIF protein is also useful as anantigen in the immunological diagnosis of bone diseases. Pharmaceuticalpreparations containing OCIF protein as an active ingredient areformulated and can be orally or parenterally administered. Thepreparation contains the OCIF protein of the present invention as aneffective ingredient and is safely administered to humans and animals.Examples of pharmaceutical preparations include compositions forinjection or intravenous drip, suppositories, nasal preparations,sublingual preparations, and tapes for percutaneous absorption. Thepharmaceutical preparation for injection can be prepared by mixing apharmacologically effective amount of OCIF protein and apharmaceutically acceptable carrier. The carriers are vehicles and/oractivators, e.g. amino acids, saccharides, cellulose derivatives, andother organic and inorganic compounds, which are generally added toactive ingredients. When the OCIF protein is mixed with the vehiclesand/or activators for injection, pH adjusters, buffers, stabilizers,solubilizing agents, etc. can be added by conventional methods, ifnecessary.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be further explained by the followingexamples, though the scope of the invention is not restricted thereto.

EXAMPLE 1

Preparation of a Conditioned Medium of Human Fibroblast IMR-90

Human fetal lung fibroblast IMR-90 (ATCC-CCL186) cells were cultured onalumina ceramic pieces (80 g) (alumina: 99.5%, manufactured by ToshibaCeramic K.K.) in DMEM medium (manufactured by Gibco BRL Co.)supplemented with 5% CS and 10 mM HEPES buffer (500 ml/roller bottle) at37° C. in the presence of 5% CO₂ for 7 to 10 days using 60 rollerbottles (490 cm², 110×171 mm, manufactured by Corning Co.) in staticculture. The conditioned medium was harvested and a fresh medium wasadded to the roller bottles. About 30 L of IMR-90 conditioned medium perbatch culture was obtained. The conditioned medium was designated assample 1.

EXAMPLE 2

Assay Method for Osteoclast Development Inhibitory Activity

Osteoclast development inhibitory activity was assayed by measuringtartrate-resistant acid phosphatase (TRAP) activity according to themethods of M. Kumegawa et al. (Protein Nucleic Acid Enzyme, vol. 34 p999, 1989) and N. Takahashi et al. (Endocrinology, vol. 122, p 1373,1988 ) with modifications. Briefly, bone marrow cells obtained from a 17day-old mouse were suspended in α-MEM™ (manufactured by GIBCO BRL Co.)containing 10% FBS, 2×10⁻⁸ M of activated vitamin D₃ and a test sampleand were inoculated into each well of a 96-well plate at a cell densityof 3×10⁵ cells/0.2 ml/well. The plates were incubated for 7 days at 37°C. in humidified 5% CO₂. Cultures were maintained by replacing 0.16 mlof old medium with the same volume of fresh medium on day 3 and day 5after cultivation began. On day 7, the plates were washed with phosphatebuffered saline, and the cells were fixed with ethanol/acetone (1:1) for1 min. at room temperature. Osteoclast development was tested bydetermining acid phosphatase activity using a kit (Acid Phosphatase,Leucocyte, Catalog No. 387-A, manufactured by Sigma Co.). A decrease inthe number of TRAP positive cells was taken as an indication of OCIFactivity.

EXAMPLE 3

Purification of OCIF

i) Heparin SEPHAROSE™ CL-6B Column Chromatography

90 L of IMR-90 conditioned medium (sample 1) was filtered using a 0.22μmembrane filter (hydrophilic MILLIDISK™ 2000 cm², Millipore Co.), andwas divided into three 30 liter portions. Each portion was applied to aheparin SEPHAROSE™ CL-6B column (5×4.1 cm, Pharmacia Co.) equilibratedwith 10 mM Tris-HCl containing 0.3 M NaCl, pH 7.5. After washing thecolumn with 10 mM Tris-HCl pH 7.5 at a flow rate of 500 ml/hr., theheparin SEPHAROSE™ CL-6B adsorbent protein fraction was eluted with 10mM Tris-HCl, pH 7.5, containing 2 M NaCl. The fraction was designatedsample 2.

ii) HILOAD™-Q/FF Column Chromatography

The heparin SEPHAROSE™-adsorbent fraction (sample 2) was dialyzedagainst 10 mM Tris-HCl, pH 7.5, supplemented with CHAPS to a finalconcentration of 0.1%, incubated at 4° C. overnight and divided into twoportions. Each portion was then applied to an anion-exchange column(HILOAD™-Q/FF, 2.6×10 cm, Pharmacia Co.) which was equilibrated with 50mM Tris-HCl, 0.1% CHAPS, pH 7.5 to obtain a non-adsorbent fraction (1000ml). The fraction was designated sample 3.

iii) HILOAD™-S/HP Column Chromatography

The HILOAD™-Q non-adsorbent fraction (sample 3) was applied to acation-exchange column (HILOAD™-S/HP, 2.6×10 cm, Pharmacia Co.) whichwas equilibrated with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5. After washingthe column with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed proteinwas eluted with a linear gradient from 0 to 1 M NaCl at a flow rate of 8ml/min for 100 min. and fractions (12 ml) were collected. Every tenfractions from numbers 1 to 40 were pooled to form one portion. 100 μLeach of the four portions was tested for OCIF activity. OCIF activitywas observed in fractions 11 to 30 (as shown in FIGS. 1A and 1B).Fractions 21 to 30, which had higher specific activity, were pooled anddesignated sample 4.

iv) Heparin-5PW Affinity Column Chromatography

One hundred and twenty ml of HILOAD™-S fractions 21 to 30 (sample 4) wasdiluted with 240 ml of 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5, and appliedto a heparin-5PW affinity column (0.8×7.5 cm, Tosoh Co.) which wasequilibrated with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5. After washing thecolumn with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein waseluted with a linear gradient from 0 to 2 M NaCl at a flow rate of 0.5ml/min for 60 min. and fractions (0.5 ml) were collected. Fifty μL wereremoved from each fraction to test for OCIF activity. The activefractions, eluted with 0.7 to 1.3 M NaCl were pooled and designatedsample 5.

v) Blue 5PW Affinity Column Chromatography

Ten ml of sample 5 were diluted with 190 ml of 50 mM Tris-HCl, 0.1%CHAPS, pH 7.5 and applied to a blue-5PW affinity column, (0.5×5 cm,Tosoh Co.) which was equilibrated with 50 mM Tris-HCl, 0.1% CHAPS, pH7.5. After washing the column with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5,the adsorbed protein was eluted with a 30 ml linear gradient from 0 to 2M NaCl at a flow rate of 0.5 ml/min. and fractions (0.5 ml) werecollected. Using 25 μL of each fraction, OCIF activity was evaluated.Fractions 49 to 70, eluted with 1.0-1.6 M NaCl, had OCIF activity.

vi) Reverse Phase Column Chromatography

The blue 5PW fraction obtained by collecting fractions 49 and 50 wasacidified with 10 μL of 25% TFA and applied to a reverse phase C4 column(BU-300, 2.1×220 mm, manufactured by Perkin-Elmer) which wasequilibrated with 0.1% of TFA and 25% acetonitrile. The adsorbed proteinwas eluted with a linear gradient from 25 to 55% acetonitrile at a flowrate of 0.2 ml/min. for 60 min., and each protein peak was collected(FIG. 3). One hundred μL of each peak fraction was tested for OCIFactivity, and peaks 6 and 7 had OCIF activity. The result was shown inTable 1.

TABLE 1 Dilution Sample 1/40 1/120 1/360 1/1080 Peak 6 ++ ++ + − Peak 7++ + − − [++ means OCIF activity inhibiting osteoclast development morethan 80%, + means OCIF activity inhibiting osteoclast developmentbetween 30% and 80%, and − means no OCIF activity.]

EXAMPLE 4

Molecular Weight of OCIF Protein

The two protein peaks with OCIF activity (peaks 6 and 7) were subjectedto SDS-polyacrylamide gel electrophoresis under reducing andnon-reducing conditions. Briefly, 20 μL of each peak fraction wasconcentrated under vacuum and dissolved in 1.5 μL of 10 mM Tris-HCl, pH8, 1 mM EDTA, 2.5% SDS, 0.01% bromophenol blue, and incubated at 37° C.overnight under non-reducing conditions or under reducing conditions(with 5% of 2-mercaptoethanol). Each 1.0 μL of sample was then analyzedby SDS-polyacrylamide gel electrophoresis with a gradient gel of 10-15%acrylamide (Pharmacia Co.) and an electrophoresis-device (Fast System,Pharmacia Co.). The following molecular weight marker proteins were usedto calculate molecular weight: phosphorylase b (94 kD), bovine serumalbumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), trypsininhibitor (20.0 kD), and lactalbumin (14.4 kD). After electrophoresis,protein bands were visualized by silver stain using Phast Silver StainKit. The results are shown in FIG. 4.

A protein band with an apparent molecular weight of 60 kD was detectedin the peak 6 sample under both reducing and non-reducing conditions. Aprotein band with an apparent 60 kD was detected under reducingconditions and a protein band with an apparent 120 kD was detected undernon-reducing conditions in the peak 7 sample. Therefore, the protein ofpeak 7 was considered to be a homodimer of the protein of peak 6.

EXAMPLE 5

Thermostability of OCIF

Twenty μL of sample from the blue-5PW fractions 51 and 52 was diluted to30 μL with 10 mM phosphate buffered saline, pH 7.2, and incubated for 10min. at 70° C. or 90° C., or for 30 min. at 56° C. The heat-treatedsamples were tested for OCIF activity. The results are shown in Table 2.

TABLE 2 Thermostability of OCIF Dilution Sample 1/300 1/900 1/2700Untreated ++ + − 70° C., 10 min + − − 56° C., 30 min + − − 90° C., 10min − − − [++ means OCIF activity inhibiting osteoclast development morethan 80%, +means OCIF activity inhibiting osteoclast development between30% and 80%, and − means no OCIF activity.]

EXAMPLE 6

Internal Amino Acid Sequence of OCIF Protein

Each 2 fractions (1 ml) from fractions 51 to 70 of the blue-5PWfractions were acidified with 10 μL of 25% TFA, and applied to a reversephase C4 column (BU-300, 2.1×220 mm, manufactured by Perkin-Elmer Co.)equilibrated with 25% acetonitrile containing 0.1% TFA. The adsorbedprotein was eluted with a 12 ml linear gradient of 25 to 55%acetonitrile at a flow rate of 0.2 ml/min, and the protein fractionscorresponding to peaks 6 and 7 were collected, respectively. The proteinfrom each peak was applied to a protein sequencer (PROCISE™ 494,Perkin-Elmer Co.). However, the N-terminal sequence of the protein ofeach peak could not be analyzed. Therefore, the N-terminus of theprotein of each peak was considered to be blocked. Internal amino acidsequences of these proteins were therefore analyzed.

The protein from peak 6 or 7 purified by C4-HPLC, was concentrated bycentrifugation and pyridilethylated under reducing conditions. Briefly,50 μL of 0.5 M Tris-HCl, pH 8.5, containing 100 μg of dithiothreitol, 10mM EDTA, 7 M guanidine-HCl, and 1% CHAPS was added to each of thesamples, and the mixtures were incubated overnight in the dark at roomtemperature. Each mixture was acidified with 25% TFA (a finalconcentration 0.1%) and applied to a reverse phase C4 column (BU300,2.1×30 mm, Perkin-Elmer Co.) equilibrated with 20% acetonitrilecontaining 0.1% TFA. The pyridil-ethylated OCIF protein was eluted witha 9 ml linear gradient from 20 to 50% acetonitrile at a flow rate of 0.3ml/min, and each protein peak was collected. The pyridil-ethylated OCIFprotein was concentrated under vacuum and dissolved in 25 μL of 0.1 MTris-HCl, pH 9, containing 8 M Urea, and 0.1% TWEEN™ 80. Seventy-threeμL of 0.1 M Tris-HCl, pH 9, and 0.02 μg of lysyl endopeptidase (WakoPure Chemical, Japan) were added to the tube, and incubated at 37° C.for 15 hours. Each digest was acidified with 1 μL of 25% TFA and wasapplied to a reverse phase C8 column (RP-300, 2.1×220 mm, Perkin-ElmerCo.) equilibrated with 0.1% TFA.

The peptide fragments were eluted from the column with a linear gradientof 0 to 50% acetonitrile at a flow rate of 0.2 ml/min for 70 min., andeach peptide peak was collected. Each peptide fragment (P1-P3) wasapplied to the protein sequencer. The sequences of the peptides areshown in SEQ ID NOs: 1-3, respectively.

EXAMPLE 7

Determination of the Nucleotide Sequence of OCIF cDNA

i) Isolation of Poly(A)+RNA from IMR-90 Cells

About 10 μg of poly(A)+RNA was isolated from 1×10⁸ cells of IMR-90 usinga FASTTRACK™ mRNA isolation kit (Invitrogen) according to themanufacturer's instructions.

ii) Preparation of Mixed Primers

The following two mixed primers were synthesized based on the amino acidsequences of two peptides (peptide P2 and peptide P3, SEQ ID NOs: 2 and3, respectively). All the oligonucleotides in the mixed primers No. 2F(SEQ ID NO: 107) can code for the amino acid sequence from the sixthresidue, glutamine (Gln) to the twelfth residue, leucine (Leu), inpeptide P2. All the oligonucleotides in the mixed primers No. 3R (SEQ IDNO: 108) can code for the amino acid sequence from the sixth residue,histidine (His), to the twelfth residue, lysine (Lys), in peptide P3.The sequences of the mixed primers No. 2F and No. 3R were shown in Table3.

TABLE 3 No. 2F (SEQ ID NO: 107)        5′-CAAGAACAAA CTTTTCAATT-3′            G  G  G   C  C  GC                          A                         G No. 3R (SEQ ID NO: 108)        5′-TTTATACATTGTAAAAGAAT G-3′           C  G     C   G  GCTG                   A      C                    G      T

iii) Amplification of an OCIF cDNA Fragment by PCR (Polymerase ChainReaction)

First strand cDNA was generated using a SUPERSCRIPT™ II cDNA synthesiskit 23 (Gibco BRL) and 1 μg of poly(A)+RNA obtained in EXAMPLE 7-i),according to the manufacturer's instructions. The DNA fragment encodingOCIF was obtained by PCR using cDNA template and the primers shown inEXAMPLE 7-ii).

PCR was performed using the following conditions:

10× Ex Taq Buffer (Takara Shuzo) 5 μl 2.5 mM solution of dNTPs 4 μl cDNAsolution 1 μl Ex Taq (Takara Shuzo) 0.25 μl sterile distilled water29.75 μl 40 μM solution of primers No. 2F 5 μl 40 μM solution of primersNo. 3R 5 μl

The components of the reaction were mixed in a microcentrifuge tube. Aninitial denaturation step at 95° C. for 3 min was followed by 30 cyclesof denaturation at 95° C. for 30 sec, annealing at 50° C. for 30 sec andextention at 70° C. for 2 min. After the amplification, a finalextention step was performed at 70° C. for 5 min. The sizes of the PCRproducts were determined on a 1.5% agarose gel electrophoresis. Anapproximately 400 bp OCIF DNA fragment was obtained.

EXAMPLE 8

Cloning of the OCIF cDNA Fragment Amplified by PCR and Determination ofits DNA Sequence

The OCIF cDNA fragment amplified by PCR in EXAMPLE 7-iii) was insertedinto the plasmid pBLUESCRIPT II SK™ using a DNA ligation kit ver. 2(Takara Shuzo) according to the method of Marchuk, D. et al. (NucleicAcids Res., vol 19, p 1154, 1991). E. coli strain DH5 α (Gibco BRL) wastransformed with the ligation mixture. The transformants were grown anda plasmid containing the OCIF cDNA (about 400 bp) was purified usingcommonly used methods. This plasmid was called pBSOCIF. The sequence ofthe OCIF cDNA in pBSOCIF was determined using a TAQ DYE DEOXY TERMINATERCYCLE SEQUENCING™ kit (Perkin Elmer). The size of the OCIF cDNA is 397bp. The OCIF cDNA encodes an amino acid sequence containing 132residues. The amino acid sequences of the internal peptides (peptide P2and peptide P3, SEQ ID NOs: 2 and 3, respectively) that were used todesign the primers were found at the amino or carboxyl terminus of the132 amino acid sequence predicted by the 397 bp OCIF cDNA. In addition,the amino acid sequence of the internal peptide P1 (SEQ ID NO: 1) wasalso found in the predicted amino acid sequence of OCIF. These data showthat the 397 bp OCIF cDNA is a portion of the full length OCIF cDNA.

EXAMPLE 9

Preparation of the DNA Probe

The 397 bp OCIF cDNA was prepared according to the conditions describedin EXAMPLE 7-iii). The OCIF cDNA was subjected to a preparative agarosegel electrophoresis. The OCIF cDNA was purified from the gel using aQIAEX™ gel extraction kit (QIAGEN), labeled with [α³²P]dCTP usingMegaprime DNA labeling system (Amersham) and used to select a phagecontaining the full length OCIF cDNA.

EXAMPLE 10

Preparation of the cDNA Library

cDNA was generated using a Great Lengths cDNA synthesis kit (Clontech),oligo (dT) primer, [α³²P]dCTP and 2.5 μg of poly(A)+RNA obtained inEXAMPLE 7-i), according to the manufacturer's instructions. AnEcoRI-SalI-NotI adaptor was ligated to the cDNA. The cDNA was separatedfrom free adaptor DNA and unincorporated free [α³²P]dCTP. The purifiedcDNA was precipitated with ethanol and dissolved in 10 μL of TE buffer(10 mM Tris-HCl (pH 8.0), 1 mM EDTA). The cDNA comprising the adaptorwas ligated into λZAP EXPRESS™ vector (Stratagene) at the EcoRI site.The recombinant λZAP EXPRESS™ phage DNA containing the cDNA was in vitropackaged using a GIGAPACK™ gold # II packaging extract (Stratagene)yielding a recombinant λZAP EXPRESS™ phage library.

EXAMPLE 11

Screening of Recombinant Phage

Recombinant phages obtained in EXAMPLE 10 were used to infect E. colistrain, XL1-Blue MRF′ (Stratagene) at 37° C. for 15 min. The infected E.coli cells were added to NZY medium containing 0.7% agar at 50° C. andplated onto NZY agar plates. After the plates were incubated at 37° C.overnight, HYBOND™ N (Amersham) membranes were placed on the surface ofthe plates containing plaques. The membranes were denatured in alkalisolution, neutralized, and washed in 2×SSC according to standardmethods. The phage DNA was immobilized onto the membranes using UVCROSSLINK™ (Stratagene). The membranes were incubated in hybridizationbuffer (Amersham) containing 100 μg/ml salmon sperm DNA at 65° C. for 4hours and then incubated at 65° C. overnight in the same buffercontaining 2×10⁵ cpm/ml of denatured OCIF DNA probe. The membranes werewashed twice with 2×SSC and twice with a solution containing 0.1×SSC and0.1% SDS at 65° C. for 10 min each time. The positive clones werepurified by repeating the screening twice. The purified λZAP EXPRESS™phage clone containing a DNA insert of about 1.6 kb was used in theexperiments described below. This phage was called λ OCIF. The purifiedλ OCIF was used to infect E. coli strain XL-1 blue MRF′ (Stratagene)according to the protocol in the λZAP EXPRESS™ cloning kit (Stratagene).The culture broth of infected XL-1 blue MRF′ was prepared. Purified λOCIF and EXASSIST™ helper phage (Stratagene) were coinfected into E.coil strain XL-1 blue MRF′, according to the protocol supplied with thekit. The culture broth of the coinfected XL-1 blue MRF′ was added to aculture of E. coli strain XLOR (Stratagene) to transform them. Thus weobtained a Kanamycin-resistant transformant harboring a plasmiddesignated pBKOCIF which is a pBKCMV (Stratagene) vector containing the1.6 kb insert fragment.

The transformant including the plasmid containing about 1.6 kb OCIF cDNAwas obtained by lifting the Kanamycin-resistant colonies. The plasmidwas called pBKOCIF. The transformant has been deposited in the NationalInstitute of Bioscience and Human-Technology (NIBH), Agency ofIndustrial Science and Technology as “FERM BP-5267” as pBK/01F10. Anational deposit (Accession number, FERM P-14998) was transferred to theinternational deposit, on Oct. 25, 1995 according to the Budapesttreaty. The transformant pBK/01F10 was grown and the plasmid pBKOCIF waspurified according to standard methods.

EXAMPLE 12

Determination of the Nucleotide Sequence of OCIF cDNA Containing theFull Coding Region.

The nucleotide sequence of OCIF cDNA obtained in EXAMPLE 11 wasdetermined using a TAQ DYE DEOXY TERMINATER CYCLE SEQUENCING™ kit(Perkin Elmer). The primers used were T3, T7 (Stratagene) and syntheticprimers designed according to the OCIF cDNA sequence. The sequences ofthese primers are shown in SEQ ID NOs: 16 to 29. The nucleotide sequenceof the OCIF cDNA is shown in SEQ ID NO: 6 and the amino acid sequencepredicted by the cDNA sequence is shown in SEQ ID NO: 5.

EXAMPLE 13

Production of Recombinant OCIF by 293/EBNA Cells

i) Construction of the Plasmid for Expressing OCIF cDNA

pBKOCIF, containing about 1.6 kb OCIF cDNA, was prepared as described inEXAMPLE 11 and digested with restriction enzymes BamHI and XhoI. TheOCIF cDNA insert was cut out, isolated by an agarose gel electrophoresisand purified using a QIAEX™ gel extraction kit (QIAGEN). The purifiedOCIF cDNA insert was ligated into the expression vector pCEP4(Invitrogen) using DNA ligation kit ver. 2 (Takara Shuzo) digested withrestriction enzymes BamHI and XhoI. E. coli strain DH5 α (Gibco BRL) wastransformed with the ligation mixture.

The transformants were grown and the plasmid containing the OCIF cDNA(about 1.6 kb) was purified using a QIAGEN™ column (QIAGEN). Theexpression plasmid pCEPOCIF was precipitated with ethanol and dissolvedin sterile distilled water for use in the experiments described below.

ii) Transient Expression of OCIF cDNA and Analysis of OCIF BiologicalActivity

Recombinant OCIF was produced using the expression plasmid pCEPOCIF(prepared in EXAMPLE 13-i) according to the method described below.8×10⁵ cells of 293/EBNA (Invitrogen) were inoculated into each well of a6-well plate using IMDM containing 10% fetal bovine serum (FBS; GibcoBRL). After the cells were incubated for 24 hours, the culture mediumwas removed and the cells were washed with serum free IMDM. Theexpression plasmid pCEPOCIF and lipofectamine (Gibco BRL) were dilutedwith OPTI-MEM™ (Gibco BRL), mixed, and added to the cells in each wellaccording to the manufacturer's instructions. Three μg of pCEPOCIF and12 μL of lipofectamine were used for each transfection. After the cellswere incubated with pCEPOCIF and lipofectamine for 38 hours, the mediumwas replaced with 1 ml of OPTI-MEM™. After incubation for 30 hours, theconditioned medium was harvested and used for the biological assay. Thebiological activity of OCIF was analyzed according to the methoddescribed below. Bone marrow cells obtained from 17 day old mice weresuspended in α-MEM™ (manufactured by GIBCO BRL Co.) containing 10% FBS,1×10⁻⁸M activated vitamin D₃ and a test sample, and were inoculated andcultured for 7 days at 37° C. in humidified 5% CO₂ as described inEXAMPLE 2. During incubation, 160 μL of old medium in each well wasreplaced with the same volume of the fresh medium containing test samplediluted with 1×10⁻⁸ M of activated vitamin D₃ and α-MEM™ containing FBSon day 3 and day 5. On day 7, after washing the wells with phosphatebuffered saline, cells were fixed with ethanol/acetone (1:1) for 1 min.and osteoclast development was tested using an acid phosphatase activitymeasuring kit (Acid Phosphatase, Leucocyte, Catalog No. 387A, SigmaCo.). A decrease in the number of TRAP positive cells was taken as anOCIF activity. The conditioned medium showed the same OCIF activity asnatural OCIF protein from IMR-90 conditioned medium (Table 4).

TABLE 4 OCIF activity of 293/EBNA conditioned medium. Dilution CulturedCell 1/20 1/40 1/80 1/160 1/320 1/640 1/1280 OCIF expression ++ ++ ++ ++++ + − vector transfected vector − − − − − − − transfected untreated − −− − − − − [++; OCIF activity inhibiting osteoclast development more than80%, +; OCIF activity inhibiting osteoclast development between 30% and80%, and −; no OCIF activity.]

iii) Isolation of Recombinant OCIF Protein from 293/EBNA-conditionedMedium

293/EBNA-conditioned medium (1.8 L) obtained by cultivating the cellsdescribed in EXAMPLE 13-ii) was supplemented with 0.1% CHAPS andfiltrated using a 0.22 μm membrane filter (Sterivex GS, Millipore Co.).The conditioned medium was applied to a 50 ml heparin SEPHAROSE™ CL-6Bcolumn (2.6×10 cm, Pharmacia Co.) equilibrated with 10 mM Tris-HCl, pH7.5. After washing the column with 10 mM Tris-HCl, pH 7.5, the adsorbedprotein was eluted from the column with a linear gradient from 0 to 2 MNaCl at a flow rate of 4 ml/min for 100 min. and 8 ml fractions werecollected. Using 150 μL of each fraction, OCIF activity was assayedaccording to the method described in EXAMPLE 2. An OCIF active 112 mlfraction, eluted with approximately 0.6 to 1.2 M NaCl, was obtained.

One hundred twelve ml of the active fraction was diluted to 1000 ml with10 mM Tris-HCl, 0.1% CHAPS, pH 7.5, and applied to a heparin affinitycolumn (heparin-5PW, 0.8×7.5 cm, Tosoh Co.) equilibrated with 10 mMTris-HCl, 0.1% CHAPS, pH 7.5. After washing the column with 10 mMTris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein was eluted from thecolumn with a linear gradient from 0 to 2 M NaCl at a flow rate of 0.5ml/min for 60 min. and 0.5 ml fractions were collected. Four μL of eachfraction was analyzed by SDS-polyacrylamide gel electrophoresis underreducing and non-reducing conditions as described in EXAMPLE 4. A singleband of rOCIF protein with an apparent molecular weight of 60 kD wasdetected in fractions from 30 to 32 by SDS-PAGE under reducingconditions. Bands of rOCIF protein with apparent molecular weights of 60kD and 120 kD were also detected in fractions from 30 to 32 undernon-reducing conditions. The isolated rOCIF from fractions 30 to 32 wasdesignated as recombinant OCIF derived from 293/EBNA (rOCIF(E)). 1.5 mlof the rOCIF(E) (535 μg/ml) was obtained when determined by the methodof Lowry, using bovine serum albumin as a standard protein.

EXAMPLE 14

Production of Recombinant OCIF Using CHO Cells

i) Construction of the Plasmid for Expressing OCIF

pBKOCIF containing about 1.6 kb OCIF cDNA was prepared as described inEXAMPLE 11, and digested with restriction enzymes SalI and EcoRV. About1.4 kb OCIF cDNA insert was separated by agarose gel electrophoresis andpurified from the gel using a QIAEX™ gel extraction kit (QIAGEN). Theexpression vector, pcDL-SR α 296 (Molecular and Cellular Biology, vol 8,p 466-472, 1988) was digested with restriction enzymes PstI and KpnI.About 3.4 kb of the expression vector fragment was cut out, separated byagarose gel electrophoresis and purified from the gel using a QIAEX™ gelextraction kit (QIAGEN). The ends of the purified OCIF cDNA insert andthe expression vector fragment were blunted using a DNA blunting kit(Takara Shuzo). The purified OCIF cDNA insert and the expression vectorfragment were ligated using a DNA ligation kit ver. 2 (Takara Shuzo). E.coli strain DH5a α (Gibco BRL) was transformed with the ligationmixture. A transformant containing the OCIF expression plasmid, pSRαOCIF was obtained.

ii) Preparation of the Expression Plasmid

The transformant containing the OCIF expression plasmid, pSR αOCIFprepared in EXAMPLE 13-i) and the transformant containing the mouse DHFRexpression plasmid, pBAdDSV shown in WO 92/01053 were grown according tostandard methods. Both plasmids were purified by alkali treatment,polyethylene glycol precipitation, and cesium chloride density gradientultra centrifugation according to the method of Maniatis et al.(Molecular Cloning, 2nd edition).

iii) Adaptation of CHOdhFr⁻ Cells to the Protein Free Medium

CHOdhFr⁻ cells (ATCC, CRL 9096) were cultured in IMDM containing 10%fetal bovine serum. The cells were adapted to EXCELL™ 301 (JRHBioscience) and then adapted to EXCELL™ PF CHO (JRH Bioscience)according to the manufacturer's instructions.

iv) Transfection of the OCIF Expression Plasmid, and the Mouse DHFRExpression Plasmid, into CHOdhFr⁻ Cells.

CHOdhFr⁻ cells prepared in EXAMPLE 14-iii) were transfected byelectroporation with pSR αOCIF and pBAdDSV prepared in EXAMPLE 14-ii).Two hundred μg of pSR αOCIF and 20 μg of pBAdDSV were dissolved understerile conditions in 0.8 ml of IMDM (Gibco BRL) containing 10% fetalbovine serum. CHOdhFr⁻ cells (2×10⁷) were suspended in 0.8 ml of thismedium. The cell suspension was transferred to a cuvette (Bio Rad) andthe cells were transfected by electroporation using a GENE PULSER™ (BioRad) under the conditions of 360 V and 960 μF. The suspension ofelectroporated cells was transferred to T-flasks (Sumitomo Bakelite)containing 10 ml of EXCELL™ PF-CHO, and incubated in the CO₂ incubatorfor 2 days. The transfected cells were then inoculated into each well ofa 96 well plate (Sumitomo Bakelite) at a density of 5000 cells/well andcultured for about 2 weeks. The transformants expressing DHFR areselected since EXCELL™ PF-CHO does not contain nucleotides and theparental cell line CHOdhFr⁻ can not grow in this medium. Most of thetransformants expressing DHFR express OCIF since the OCIF expressionplasmid was used ten times as much as the mouse DHFR expression plasmid.The transformants whose conditioned medium had high OCIF activity wereselected from among the transformants expressing DHFR according to themethod described in EXAMPLE 2. The transformants that express largeamounts of OCIF were cloned by the limiting dilution method. The cloneswhose conditioned medium had high OCIF activity were selected asdescribed above and a transformant expressing large amounts of OCIFnamed, 5561, was obtained.

v) Production of Recombinant OCIF

To produce recombinant OCIF (rOCIF), clone 5561 was inoculated into a 3L spinner flask with EXCELL™ 301 medium (3 L) at a cell density of 1×10⁵cells/ml. The 5561 cells were cultured in a spinner flask at 37° C. for4 to 5 days. When the concentration of the 5561 cells reached 1×10⁶cells/ml, about 2.7 L of the conditioned medium was harvested. Thenabout 2.7 L of EXCELL™ 301 was added to the spinner flask and the 5561cells were cultured repeatedly.

About 20 L of the conditioned medium was harvested using the threespinner flasks.

vi) Isolation of Recombinant OCIF Protein from CHO Cell-conditionedMedium

CHO cell-conditioned medium (1.0 L) described in EXAMPLE 14-v) wassupplemented with 1.0 g CHAPS and filtrated with a 0.22 μm membranefilter (Sterivex-GS, Millipore Co.). The conditioned medium was appliedto a heparin SEPHAROSE™-FF column (2.6×10 cm, Pharmacia Co.)equilibrated with 10 mM Tris-HCl, pH 7.5. After washing the column with10 mM Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein was eluted fromthe column with a linear gradient from 0 to 2 M NaCl at a flow rate of 4ml/min for 100 min. and 8 ml fractions were collected. Using 150 μL ofeach fraction, OCIF activity was assayed according to the methoddescribed in EXAMPLE 2. An active fraction (112 ml) eluted withapproximately 0.6 to 1.2 M NaCl was obtained.

The 112 ml active fraction was diluted to 1200 ml with 10 mM Tris-HCl,0.1% CHAPS, pH 7.5, and applied to an affinity column (Blue-5PW, 0.5×5.0cm, Tosoh Co.) equilibrated with 10 mM Tris-HCl, 0.1% CHAPS, pH 7.5.After washing the column with 10 mM Tris-HCl, 0.1% CHAPS, pH 7.5, theadsorbed protein was eluted from the column with a linear gradient from0 to 3 M NaCl at a flow rate of 0.5 ml/min for 60 min. and fractions(0.5 ml) were collected. Four μL of each fraction were subjected toSDS-polyacrylamide gel electrophoresis under reducing and non-reducingconditions as described in EXAMPLE 4. A single band of rOCIF proteinwith an apparent molecular weight of 60 kD was detected in fractions 30to 38 using SDS-PAGE under reducing conditions. Bands of rOCIF proteinwith apparent molecular weights of 60 kD and 120 kD using SDS-PAGE undernon-reducing conditions were also detected in fractions 30 to 38. Theisolated rOCIF fraction, from fractions 30 to 38, was designated aspurified recombinant OCIF derived from CHO cells (rOCIF(C)). 4.5 ml ofthe rOCIF(C) (113 μg/ml) was obtained, as determined by the method ofLowry using bovine serum albumin as a standard protein.

EXAMPLE 15

Determination of N-terminal Amino Acid Sequence of rOCIFs

Three μg of the isolated rOCIF(E) and rOCIF(C) were adsorbed topolyvinylidene difluoride (PVDF) membranes with PROSPIN™ (PERKIN ELMERCo.). The membranes were washed with 20% ethanol and the N-terminalamino acid sequences of the adsorbed proteins were analyzed by proteinsequencer (PROCISE™ 492, PERKIN ELMER Co.). The determined N-terminalamino acid sequence is shown in SEQ ID NO: 7.

The N-terminal amino acid of rOCIF(E) and rOCIF(C) was glutamic acidlocated at position 22 from Met of the translation start site, as shownin SEQ ID NO: 5. The 21 amino acids from Met to Gln were identified as asignal peptide. The N-terminal amino acid sequence of OCIF isolated fromIMR-90 conditioned medium could not be determined. Accordingly, theN-terminal glutamic acid of OCIF may be blocked by the conversion ofglutamic acid to pyroglutamine within cell culture or purificationsteps.

EXAMPLE 16

Biological Activity of Recombinant (r) OCIF and Natural (n) OCIF

i) Inhibition of Vitamin D₃ Induced Osteoclast Formation in Murine BoneMarrow Cells

Each of the rOCIF(E) and nOCIF samples were diluted with α-MEM™ (GIBCOBRL Co.) containing 10% FBS and 2×10⁻⁸ M of activated vitamin D₃ (afinal concentration of 250 ng/ml). Each sample was serially diluted withthe same medium, and 100 μL of each diluted sample was added to eachwell of a 96-well plate. Bone marrow cells obtained from 17 day old micewere inoculated at a cell density of 3×10⁵ cells/100 μL/well into eachwell of a 96-well plate and cultured for 7 days at 37° C. in humidified5% CO₂. On day 7, the cells were fixed and stained with an acidphosphatase measuring kit (Acid Phosphatase, Leucocyte, No. 387-A,Sigma) according to the method described in EXAMPLE 2. A decrease inacid phosphatase activity (TRAP) was taken as an indication of OCIFactivity. A decrease in acid phosphatase-positive cells was evaluated bysolubilizing the pigment of dye and measuring absorbance. Briefly, 100μL of a mixture of 0.1 N NaOH and dimethylsulfoxide (1:1) was added toeach well and the well was vibrated to solubilize the dye. Aftersolubilizing the dye completely, an absorbance of each well was measuredat 590 nm, subtracting the absorbance at 490 nm using a microplatereader (IMMUNOREADER™ NJ-2000, InterMed). The microplate reader wasadjusted to 0 absorbance using a well with monolayered bone marrow cellswhich were cultured in the medium without activated vitamin D₃. Adecrease in TRAP activity was expressed as a percentage of the controlabsorbance value (=100%) (measured for wells with bone marrow cellscultured in the absence of OCIF). The results are shown in Table 5.

TABLE 5 Inhibition of vitamin D3-induced osteoclast formation frommurine bone marrow cells OCIF concentration(ng/ml) 250 125 63 31 16 0rOCIF(E) 0 0 3 62 80 100 nOCIF 0 0 27 27 75 100 (%)

Both nOCIF and rOCIF(E) inhibited osteoclast formation in a dosedependent manner at concentrations of 16 ng/ml or greater.

ii) Inhibition of Vitamin D₃-induced Osteoclast Formation in Co-culturesof Stromal Cells and Mouse Spleen Cells.

The effect of OCIF on osteoclast formation induced by Vitamin D₃ inco-cultures of stromal cells and mouse spleen cells was tested accordingto the method of N. Udagawa et al. (Endocrinology, vol. 125, p1805-1813, 1989). Briefly, samples of each of rOCIF(E), rOCIF(C), andnOCIF were serially diluted with α-MEM™ (GIBCO BRL Co.) containing 10%FBS, 2×10⁻⁸ M activated vitamin D₃ and 2×10⁻⁷ M dexamethasone and 100 μLof each of the diluted samples was added to each well of 96well-microwell plates. Murine bone marrow-derived stromal ST2 cells(RIKEN Cell Bank RCB0224) at 5×10³ cells per 100 μL of α-MEM™ containing10% FBS and spleen cells from 8 week old ddy mice at 1×10⁵ cells per 100μL in the same medium, were inoculated into each well of a 96-well plateand cultured for 5 days at 37° C. in humidified 5% CO₂. On day 5, thecells were fixed and stained using an acid phosphatase kit (AcidPhosphatase, Leucocyte, No. 387-A, Sigma). A decrease in acidphosphatase-positive cells was taken as an indication of OCIF activity.The decrease in acid phosphatase-positive cells was evaluated accordingto the method described in EXAMPLE 16-i). The results are shown in Table6 (rOCIF(E) and rOCIF(C)) and Table 7 (rOCIF(E) and nOCIF).

TABLE 6 Inhibition of osteoclast formation in co-cultures of stromalcells and mouse spleen cells. OCIF concentration(ng/ml) 50 25 13 6 0rOCIF(E) 3 22 83 80 100 rOCIF(C) 13 19 70 96 100 (%)

TABLE 7 Inhibition of osteoclast formation in co-cultures of stromalcells and mouse spleen cells. OCIF concentration(ng/ml) 250 63 16 0rOCIF(E) 7 27 37 100 rOCIF(C) 13 23 40 100 (%)

nOCIF, rOCIF(E) and rOCIF(C) inhibited osteoclast formation in a dosedependent manner at concentrations of 6-16 ng/ml or greater.

iii) Inhibition of PTH-induced Osteoclast Formation in Murine BoneMarrow Cells.

The effect of OCIF on osteoclast formation induced by PTH was testedaccording to the method of N. Takahashi et al. (Endocrinology, vol. 122,p 1373-1382, 1988). Briefly, samples of each of rOCIF(E) and nOCIF (125ng/ml) were serially diluted with α-MEM™ (manufactured by GIBCO BRL Co.)containing 10% FBS and 2×10⁻⁸ M PTH, and 100 μL of each of the dilutedsamples was added to the wells of 96 well-plates. Bone marrow cells from17 day old ddy mice at a cell density of 3×10⁵ cells per 100 μL ofα-MEM™ containing 10% FBS were inoculated into each well of a 96-wellplate and cultured for 5 days at 37° C. in humidified 5% CO₂. On day 5,the cells were fixed with ethanol/acetone (1:1) for 1 min. at roomtemperature and stained with an acid phosphatase kit (Acid Phosphatase,Leucocyte, No. 387-A, Sigma) according to the method described inEXAMPLE 2. A decrease in acid phosphatase-positive cells was taken as anindication of OCIF activity. The decrease in acid phosphatase-positivecells was evaluated according to the method described in EXAMPLE 16-i).The results are shown in Table 8.

TABLE 8 Inhibition of PTH-induced osteoclast formation from murine bonemarrow cells. OCIF concentration(ng/ml) 125 63 31 16 8 0 rOCIF(E) 6 5858 53 88 100 nOCIF 18 47 53 56 91 100

nOCIF and rOCIF(E) inhibited osteoclast formation in a dose dependentmanner at concentrations of 16 ng/ml or greater.

iv) Inhibition of IL-11-induced Osteoclast Formation

The effect of OCIF on osteoclast formation induced by IL-11 was testedaccording to the method of T. Tamura et al. (Proc. Natl. Acad. Sci. USA,vol. 90, p 11924-11928, 1993). Briefly, samples of each of rOCIF(E) andnOCIF were serially diluted with α-MEM™ (GIBCO BRL Co.) containing 10%FBS and 20 ng/ml IL-11 and 100 μl of each diluted sample was added toeach well in a 96-well plate. Newborn mouse calvaria-derivedpre-adipocyte MC3T3-G2/PA6 cells (RIKEN Cell Bank RCB1127) at 5×10³cells per 100 μl of α-MEM™ containing 10% FBS, and spleen cells from 8week old ddy mouse, at 1×10⁵ cells per 100 μL in the same medium, wereinoculated into each well of a 96-well plate and cultured for 5 days at37° C. in humidified 5% CO₂. On day 5, the cells were fixed and stainedwith an acid phosphatase kit (Acid Phosphatase, Leucocyte, No. 387-A,Sigma). Acid phosphatase positive cells were counted under a microscopeand a decrease of the cell numbers was taken as an indication of OCIFactivity. The results are shown in Table 9.

TABLE 9 OCIF concentration(ng/ml) 500 125 31 7.8 2.0 0.5 0 nOCIF 0 0 1 413 49 31 rOCIF(E) 0 0 1 3 10 37 31

Both nOCIF and rOCIF(E) inhibited osteoclast formation in a dosedependent manner at concentrations of 2 ng/ml or greater.

The results shown in Tables 4-8 indicated that OCIF inhibits all thevitamin D₃, PTH, and IL-11-induced osteoclast formations at almost thesame doses. Accordingly, OCIF could be used for treating different typesof bone disorders due to decreased bone mass, that are caused bydifferent substances that induce bone resorption.

EXAMPLE 17

Isolation of Monomer-type OCIF and Dimer-type OCIF

Each rOCIF(E) and rOCIF(C) sample containing 100 μg of OCIF protein, wassupplemented with 1/100 volume of 25% trifluoro acetic acid and appliedto a reverse phase column (PROTEIN-RP™, 2.0×250 mm, YMC Co.)equilibrated with 30% acetonitrile containing 0.1% trifluoro aceticacid. OCIF protein was eluted from the column with a linear gradientfrom 30 to 55% acetonitrile at a flow rate of 0.2 ml/min for 50 min. andeach OCIF peak was collected. The monomer-type OCIF peak fraction anddimer-type OCIF peak fraction were each lyophilized.

EXAMPLE 18

Determination of the Molecular Weight of Recombinant OCIFs

Each 1 μg of the isolated monomer-type and dimer-type nOCIF purifiedusing a reverse phase column according to EXAMPLE 3-iv) and each 1 μg ofmonomer-type and dimer-type rOCIF described in EXAMPLE 17 wasconcentrated under vacuum. Each sample was incubated in the buffer forSDS-PAGE, subjected to SDS-polyacrylamide gel electrophoresis, andprotein bands on the gel were stained with silver according to themethod described in EXAMPLE 4. Results of electrophoresis undernon-reducing conditions and reducing conditions are shown in FIGS. 6 and7, respectively.

A protein band with an apparent molecular weight of 60 kD was detectedin each monomer-type OCIF sample, and a protein band with an apparentmolecular weight of 120 kD was detected in each dimer-type OCIF sampleunder non-reducing conditions. A protein band with an apparent molecularweight of 60 kD was detected in each monomer-type OCIF sample underreducing conditions. Accordingly, the molecular weights of monomer-typenOCIF from IMR-90 cells, rOCIF from 293/EBNA cells and rOCIF from CHOcells were almost the same (60 kD). Molecular weights of dimer-typenOCIF from IMR-90 cells, rOCIF from 293/EBNA cells, and rOCIF from CHOcells were also the same (120 kD).

EXAMPLE 19

Removal of the N-linked Oligosaccharide Chain and Measuring theMolecular Weight of Natural and Recombinant OCIF

Each sample containing 5 μg of the isolated monomer-type and dimer-typenOCIF purified using a reverse phase column according to EXAMPLE 3-iv)and each sample containing 5 μg of monomer-type and dimer-type rOCIFdescribed in EXAMPLE 17 were concentrated under vacuum. Each sample wasdissolved in 9.5 μL of 50 mM sodium phosphate buffer, pH 8.6, containing100 mM 2-mercaptoethanol, supplemented with 0.5 μL of 250 U/mlN-glycanase (Seikagaku kogyo Co.) and incubated for one day at 37° C.Each sample was supplemented with 10 μL of 20 mM Tris-HCl, pH 8.0containing 2 mM EDTA, 5% SDS, and 0.02% bromo-phenol blue and heated for5 min at 100° C. Each 1 μL of the samples was subjected toSDS-polyacrylamide gel electrophoresis, and protein bands on the gelwere stained with silver as described in EXAMPLE 4. The patterns ofelectrophoresis are shown in FIG. 8.

An apparent molecular weight of each of the deglycosylated nOCIF fromIMR-90 cells, rOCIF from CHO cells, and rOCIF from 293/EBNA cells was 40kD under reducing conditions. An apparent molecular weight of each ofthe untreated nOCIF from IMR-90 cells, rOCIF from 293/EBNA cells, andrOCIF from CHO cells was 60 kD under reducing conditions. Accordingly,the results indicate that the OCIF proteins are glycoproteins withN-linked sugar chains.

EXAMPLE 20

Cloning of OCIF Variant cDNAs and Determination of their DNA Sequences

The plasmid pBKOCIF, comprising OCIF cDNA inserted into plasmid pBKCMV(Stratagene), was obtained as in EXAMPLES 10 and 11. Further, during thescreening of the cDNA library with the 397 bp OCIF cDNA probe, thetransformants containing plasmids whose insert sizes were different fromthat of pBKOCIF were obtained. These transformants containing theplasmids were grown and the plasmids were purified according to thestandard method. The sequence of the insert DNA in each plasmid wasdetermined using a TAQ DYE DEOXY TERMINATER CYCLE SEQUENCING™ kit(Perkin Elmer). The primers used were T3, T7, (Stratagene) and syntheticprimers prepared based on the nucleotide sequence of OCIF cDNA. Thereare four OCIF variants (OCIF2, 3, 4, and 5) in addition to OCIF. Thenucleotide sequence of OCIF2 is shown in SEQ ID NO: 8 and the amino acidsequence of OCIF2 predicted by the nucleotide sequence is shown in SEQID NO: 9. The nucleotide sequence of OCIF3 is shown in SEQ ID NO: 10 andthe amino acid sequence of OCIF3 predicted by the nucleotide sequence isshown in SEQ ID NO: 11. The nucleotide sequence of OCIF4 is shown in SEQID NO: 12 and the amino acid sequence of OCIF4 predicted by thenucleotide sequence is shown in SEQ ID NO: 13. The nucleotide sequenceof OCIF5 is shown in SEQ ID NO: 14 and the amino acid sequence of OCIF5predicted by the nucleotide sequence is shown in SEQ ID NO: 15. Thestructures of OCIF variants are shown in FIGS. 9 to 12 and are brieflydescribed below.

OCIF2

The OCIF2 cDNA has a deletion of 21 bp from guanine at nucleotide number265 to guanine at nucleotide number 285 in the OCIF cDNA (SEQ ID NO: 6).

Accordingly, OCIF2 has a deletion of 7 amino acids from glutamic acid(Glu) at amino acid number 89 to glutamine (Gln) at amino acid number 95in OCIF (SEQ ID NO: 5).

OCIF3

The OCIF3 cDNA has a point mutation at nucleotide number 9 in the OCIFcDNA (SEQ ID NO: 6) where cytidine is replaced with guanine.Accordingly, OCIF3 has a mutation where asparagine (Asn) at amino acidnumber 3 in OCIF (SEQ ID NO: 5) is replaced with lysine (Lys). Themutation seems to be located in the signal sequence and has no essentialeffect on the secretion of OCIF3. OCIF3 cDNA has a deletion of 117 bpfrom guanine at nucleotide number 872 to cytidine at nucleotide number988 in the OCIF cDNA (SEQ ID NO: 6).

Accordingly, OCIF3 has a deletion of 39 amino acids from threonine (Thr)at amino acid number 291 to leucine (Leu) at amino acid number 329 inOCIF (SEQ ID NO: 5).

OCIF4

The OCIF4 cDNA has two point mutations in the OCIF cDNA (SEQ ID NO: 6).Cytidine at nucleotide number 9 is replaced with guanine and guanine atnucleotide number 22 is replaced with thymidine in the OCIF cDNA (SEQ IDNO: 6).

Accordingly, OCIF4 has two mutations. Asparagine (Asn) at amino acidnumber 3 in OCIF (SEQ ID NO: 5) is replaced with lysine (Lys), andalanine (Ala) at amino acid number 8 is replaced with serine (Ser).These mutations seem to be located in the signal sequence and have noessential effect on the secreted OCIF4.

The OCIF4 cDNA has about 4 kb DNA, comprising intron 2 of the OCIF gene,inserted between nucleotide number 400 and nucleotide number 401 in theOCIF cDNA (SEQ ID NO: 6). The open reading frame stops in intron 2.

Accordingly, OCIF4 has an additional novel amino acid sequencecontaining 21 amino acids after alanine (Ala) at amino acid number 133in OCIF (SEQ ID NO: 5).

OCIF5

The OCIF5 cDNA has a point mutation at nucleotide number 9 in the OCIFcDNA (SEQ ID NO: 6) where cytidine is replaced with guanine.

Accordingly, OCIF5 has a mutation where asparagine (Asn) at amino acidnumber 3 in OCIF (SEQ ID NO: 5) is replaced with lysine (Lys). Themutation seems to be located in the signal sequence and has no essentialeffect on the secretion of OCIF5.

The OCIF5 cDNA has the latter portion (about 1.8 kb) of intron 2 betweennucleotide number 400 and nucleotide number 401 in OCIF cDNA (SEQ ID NO:6). The open reading frame stops in the latter portion of intron 2.

Accordingly, OCIF5 has an additional novel amino acid sequencecontaining 12 amino acids after alanine (Ala) at amino acid number 133in OCIF (SEQ ID NO: 5).

EXAMPLE 21

Production of OCIF Variants

i) Construction of the Plasmid for Expressing OCIF Variants

Plasmids containing OCIF2 or OCIF3 cDNA were obtained as described inEXAMPLE 20 and called pBKOCIF2 and pBKOCIF3, respectively. pBKOCIF2 andpBKOCIF3 were digested with restriction enzymes BamHI and XhoI. TheOCIF2 and OCIF3 cDNA inserts were separated by agarose gelelectrophoresis and purified from the gel using a QIAEX™ gel extractionkit (QIAGEN). The purified OCIF2 and OCIF3 cDNA inserts wereindividually ligated using a DNA ligation kit ver. 2 (Takara Shuzo) tothe expression vector pCEP4 (Invitrogen) that had been digested withrestriction enzymes BamHI and XhoI. E. coli strain DH5 α (Gibco BRL) wastransformed with the ligation mixture.

The plasmid containing OCIF4 cDNA was obtained as described in EXAMPLE20 and called pBKOCIF4. pBKOCIF4 was digested with restriction enzymesSpeI and XhoI (Takara Shuzo). The OCIF4 cDNA insert was separated byagarose gel electrophoresis, and purified from the gel using a QIAEX™gel extraction kit (QIAGEN). The purified OCIF4 cDNA insert was ligatedusing a DNA ligation kit ver. 2 (Takara Shuzo) to an expression vectorpCEP4 (Invitrogen) that had been digested with restriction enzymes NheIand XhoI (Takara Shuzo). E. coli strain DH5 α (Gibco BRL) wastransformed with the ligation mixture.

The plasmid containing OCIF5 cDNA was obtained as described in EXAMPLE20 and called pBKOCIF5. pBKOCIF5 was digested with the restrictionenzyme HindIII (Takara Shuzo). The 5′ portion of the coding region inthe OCIF5 cDNA insert was separated by agarose gel electrophoresis andpurified from the gel using QIAEX™ gel extraction kit (QIAGEN). The OCIFexpression plasmid, pCEPOCIF, obtained in EXAMPLE 13-i) was digestedwith the restriction enzyme HindIII (Takara Shuzo). The 5′ portion ofthe coding region in the OCIF cDNA was removed. The rest of the plasmidthat contains pCEP vector and the 3′ portion of the coding region ofOCIF cDNA was called pCEPOCIF-3′. pCEPOCIF3′ was separated by agarosegel electrophoresis and purified from the gel using a QIAEX™ gelextraction kit (QIAGEN). The OCIF5 cDNA HindIII fragment and pCEPOCIF-3′were ligated using a DNA ligation kit ver. 2 (Takara Shuzo). E. colistrain DH5 α (Gibco BRL) was transformed with the ligation mixture.

The transformants obtained were grown at 37° C. overnight and the OCIFvariant expression plasmids (pCEPOCIF2, pCEPOCIF3, pCEPOCIF4, andpCEPOCIF5) were purified using QIAGEN™ columns (QIAGEN). TheseOCIF-variant-expression plasmids were precipitated with ethanol,dissolved in sterile distilled water, and used in the experimentsdescribed below.

ii) Transient Expression of OCIF Variant cDNAs and Analysis of theBiological Activity of Recombinant OCIF Variants.

Recombinant OCIF variants were produced using the expression plasmids,pCEPOCIF2, pCEPOCIF3, pCEPOCIF4, and pCEPOCIF5 as described in EXAMPLE21-i) according to the method described in EXAMPLE 13-ii). Thebiological activities of recombinant OCIF variants were analyzed. Theresults were that these OCIF variants (OCIF2, OCIF3, OCIF4, and OCIF5)had weak activity.

EXAMPLE 22

Preparation of OCIF Mutants

i) Construction of a Plasmid Vector for Subcloning cDNAs Encoding OCIFMutants

The plasmid vector (5 μg) described in EXAMPLE 11 was digested withrestriction enzymes BamHI and XhoI (Takara Shuzo). The digested DNA wassubjected to preparative agarose gel electrophoresis. A DNA fragmentwith an approximate size of 1.6 kilobase pairs (kb) that contained theentire coding sequence for OCIF was purified from the gel using a QIAEX™gel extraction kit (QIAGEN). The purified DNA was dissolved in 20 μL ofsterile distilled water. This solution was designated DNA solution 1.pBLUESCRIPT II SK+™ (3 μg) (Stratagene) was digested with restrictionenzymes BamHI and XhoI (Takara Shuzo). The digested DNA was subjected topreparative agarose gel electrophoresis. A DNA fragment with anapproximate size of 3.0 kb was purified from the gel using a QIAEX™ DNAextraction kit (QIAGEN). The purified DNA was dissolved in 20 μL ofsterile distilled water. This solution was designated DNA solution 2.One microliter of DNA solution 2, 4 μL of DNA solution 1 and 5 μL ofligation buffer I from a DNA ligation kit ver. 2 (Takara Shuzo) weremixed and incubated at 16° C. for 30 min. (The ligation mixture was usedin the transformation of E. coli in the manner described below).Conditions for transformation of E. coli were as follows. One hundredmicroliters of competent E. coli strain DH5 α cells (GIBCO BRL) and 5 μLof the ligation mixture were mixed in a sterile 15-ml tube (IWAKIglass). The tube was kept on ice for 30 min. After incubation for 45 secat 42° C., 250 μL of L broth (1% Tryptone, 0.5% yeast extract, 1% NaCl)was added to the cells. The cell suspension was then incubated for 1 hr.at 37° C. with shaking. Fifty microliters of the cell suspension wasplated onto an L-agar plate containing 5 μg/ml of ampicillin. The platewas incubated overnight at 37° C.

Six colonies which grew on the plate were each incubated in 2 ml ofL-broth containing 50 μg/ml ampicillin overnight at 37° C. with shaking.The structure of the plasmids in the colonies was analyzed. A plasmid inwhich the 1.6-kb DNA fragment containing the entire OCIF cDNA isinserted between the digestion sites of BamHI and XhoI of pBLUESCRIPT IISK+™ was obtained and designated as pSK+-OCIF.

ii) Preparation of Mutants in which One of the Cys Residues in OCIF isReplaced with a Ser Residue

1) Introduction of Mutations into OCIF cDNA

OCIF mutants were prepared in which one of the five Cys residues presentin OCIF at positions 174, 181, 256, 298 and 379 (in SEQ ID NO: 4) wasreplaced with a Ser residue and were designated OCIF-C19S (174Cys toSer), OCIF-C20S (181Cys to Ser), OCIF-C21S (256Cys to Ser), OCIF-C22S(298Cys to Ser) and OCIF-C23S (379Cys to Ser), respectively. The aminoacid sequences of these mutants are provided in the sequence listing asSEQ ID NOs: 62, 63, 64, 65, and 66, respectively.

To prepare the mutants, nucleotides encoding the corresponding Cysresidues were replaced with those encoding Ser. Mutagenesis was carriedout by a two-step polymerase chain reaction (PCR). The first step of thePCRs consisted of two reactions, PCR 1 and PCR 2.

PCR 1 10× Ex Taq Buffer (Takara Shuzo) 10 μl 2.5 mM solution of dNTPs 8μl the plasmid vector described in 2 μl EXAMPLE 11 (8 ng/ml) steriledistilled water 73.5 μl 20 μM solution of primer 1 5 μl 100 μM solutionof primer 2 1 μl (for mutagenesis) Ex Taq (Takara Shuzo) 0.5 μl PCR 210× Ex Taq Buffer (Takara Shuzo) 10 μl 2.5 mM solution of dNTPs 8 μl theplasmid vector described in 2 μl EXAMPLE 11 (8 ng/ml) sterile distilledwater 73.5 μl 20 μM solution of primer 3 5 μl 100 μM solution of primer4 1 μl (for mutagenesis) Ex Taq (Takara Shuzo) 0.5 μl

Specific sets of primers were used for each mutation and othercomponents were unchanged. Primers used for the reactions are shown inTable 10. The nucleotide sequences of the primers are shown in SEQ IDNOs: 20, 23, 27 and 30-40. The PCRs were performed under the followingconditions. An initial denaturation step at 97° C. for 3 min wasfollowed by 25 cycles of denaturation at 95° C. for 1 min, annealing at55° C. for 1 min and extension at 72° C. for 3 min. After theseamplification cycles, final extension was performed at 70° C. for 5 min.The sizes of the PCR products were confirmed by agarose gelelectrophoresis of the reaction solutions. After the first PCR, excessprimers were removed using an Amicon MICROCON™ (Amicon). The finalvolume of the solutions that contained the PCR products were made to 50μL with sterile distilled water. These purified PCR products were usedfor the second PCR (PCR 3).

PCR 3 10× Ex Taq Buffer (Takara Shuzo) 10 μl 2.5 mM solution of DNTPs 8μl solution containing DNA 5 μl fragment obtained from PCR 1 solutioncontaining DNA 5 μl fragment obtained from PCR 2 sterile distilled water61.5 μl 20 μM solution of primer 1 5 μl 20 μM solution of primer 3 5 μlEx Taq (Takara Shuzo) 0.5 μl

TABLE 10 mutants primer-1 primer-2 primer-3 primer-4 OCIF-C19S IF 10C19SR IF 3 C19SF OCIF-C20S IF 10 C20SR IF 3 C20SF OCIF-C21S IF 10 C21SRIF 3 C21SF OCIF-C22S IF 10 C22SR IF 14 C22SF OCIF-C23S IF 6 C23SR IF 14C23SF

The reaction conditions were exactly the same as those for PCR 1 or PCR2. The sizes of the PCR products were confirmed by 1.0% or 1.5% agarosegel electrophoresis. The DNA fragments were precipitated with ethanol,dried under vacuum and dissolved in 40 μL of sterile distilled water.The solutions containing DNA fragments with mutations C19S, C20S, C21S,C22S and C23S were designated as DNA solution A, DNA solution B, DNAsolution C, DNA solution D and DNA solution E, respectively.

The DNA fragment which is contained in solution A (20 μL) was digestedwith restriction enzymes NdeI and SphI (Takara Shuzo). A DNA fragmentwith an approximate size of 400 base pairs (bp) was extracted from apreparative agarose gel and dissolved in 20 μL of sterile distilledwater. This DNA solution was designated DNA solution 3. Two microgramsof pSK+-OCIF were digested with restriction enzymes NdeI and SphI. A DNAfragment with an approximate size of 4.2 kb was purified from apreparative agarose gel using a QIAEX™ gel extraction kit and dissolvedin 20 μL of sterile distilled water. This DNA solution was designatedDNA solution 4. Two microliters of DNA solution 3, 3 μL of DNA solution4 and 5 μL of ligation buffer I from a DNA ligation kit ver. 2 weremixed and the ligation reaction was carried out. Competent E. colistrain DH5α cells were transformed with 5 μL of the ligation mixture.Ampicillin-resistant transformants were screened for a clone containingplasmid DNA. DNA structure was analyzed by restriction enzyme mappingand by DNA sequencing. The plasmid thus obtained was namedpSK-OCIF-C19S.

The DNA fragment contained in solution B (20 μL) was digested withrestriction enzymes NdeI and SphI. A DNA fragment with an approximatesize of 400 bp was extracted from a preparative agarose gel using aQIAEX™ gel extraction kit and dissolved in 20 μL of sterile distilledwater. This DNA solution was designated DNA solution 5. Two microlitersof DNA solution 5, 3 μL of DNA solution 4 and 5 μL of ligation buffer Ifrom a DNA ligation kit ver. 2 were mixed and the ligation reaction wascarried out. Competent E. coli strain DH5 α cells were transformed with5 μL of the ligation mixture. Ampicillin-resistant transformants werescreened for a clone containing plasmid DNA. DNA structure was analyzedby restriction enzyme mapping and by DNA sequencing. The plasmid thusobtained was named pSK-OCIF-C20S.

The DNA fragment which is contained in solution C (20 μL) was digestedwith restriction enzymes NdeI and SphI. A DNA fragment with anapproximate size of 400 bp was extracted from a preparative agarose gelusing a QIAEX™ gel extraction kit and dissolved in 20 μL of steriledistilled water. This DNA solution was designated DNA solution 6. Twomicroliters of DNA solution 6, 3 μL of DNA solution 4 and 5 μL ofligation buffer I from a ligation kit ver. 2 were mixed and the ligationreaction was carried out. Competent E. coli strain DH5 α cells weretransformed with 5 μL of the ligation mixture. Ampicillin-resistanttransformants were screened for a clone containing plasmid DNA. DNAstructure was analyzed by restriction enzyme mapping and by DNAsequencing. The plasmid thus obtained was named pSK-OCIF-C21S.

The DNA fragment which is contained in solution D (20 μL) was digestedwith restriction enzymes NdeI and BstPI. A DNA fragment with anapproximate size of 600 bp was extracted from a preparative agarose gelusing a QIAEX™ gel extraction kit and dissolved in 20 μL of steriledistilled water. This DNA solution was designated DNA solution 7. Twomicrograms of pSK+-OCIF were digested with restriction enzymes NdeI andBstPI. A DNA fragment with an approximate size of 4.0 kb was extractedfrom a preparative agarose gel using a QIAEX™ gel extraction kit anddissolved in 20 μL of sterile distilled water. This DNA solution wasdesignated DNA solution 8. Two microliters of DNA solution 7, 3 μL ofDNA solution 8 and 5 μL of ligation buffer I from a DNA ligation kitver. 2 were mixed and the ligation reaction was carried out. CompetentE. Coli strain DH5 α cells were transformed with 5 μL of the ligationmixture. Ampicillin-resistant transformants were screened for a clonecontaining plasmid DNA in which the 600-bp NdeI-BstPI fragment with themutation (the C22S mutation) is substituted for the 600-bp NdeI-BstPIfragment of pSK+-OCIF by analyzing the DNA structure. DNA structure wasanalyzed by restriction enzyme mapping and by DNA sequencing. Theplasmid thus obtained was named pSK-OCIF-C22S.

The DNA fragment which is contained in solution E (20 μL) was digestedwith restriction enzymes BstPI and EcoRV. A DNA fragment with anapproximate size of 120 bp was extracted from a preparative agarose gelusing a QIAEX™ gel extraction kit and dissolved in 20 μL of steriledistilled water. This DNA solution was designated DNA solution 9. Twomicrograms of pSK+-OCIF were digested with restriction enzymes BstEIIand EcoRV. A DNA fragment with an approximate size of 4.5 kb wasextracted from a preparative agarose gel using a QIAEX™ gel extractionkit and dissolved in 20 μL of sterile distilled water. This DNA solutionwas designated DNA solution 10. Two microliters of DNA solution 9, 3 μLof DNA solution 10 and 5 μL of ligation buffer I from a DNA ligation kitver. 2 were mixed and the ligation was carried out. Competent E. colistrain DH5 α cells were transformed with 5 μL of the ligation mixture.Ampicillin-resistant transformants were screened for a clone containingplasmid DNA. DNA structure was analyzed by restriction enzyme mappingand by DNA sequencing. The plasmid thus obtained was namedpSK-OCIF-C23S.

2) Construction of Vectors for Expressing the OCIF Mutants

pSK-OCIF-C19S, pSK-OCIF-C20S, pSK-OCIF-C21S, pSK-OCIF-C22S andpSK-OCIF-C23S were digested with restriction enzymes BamHI and XhoI. The1.6 kb BamHI-XhoI DNA fragment encoding each OCIF mutant was isolatedand dissolved in 20 μL of sterile distilled water. The DNA solutionsthat contain 1.6 kb cDNA fragments derived from pSK-OCIF-C19S,pSK-OCIF-C20S, pSK-OCIF-C21S, pSK-OCIF-C22S and pSK-OCIF-C23S weredesignated C19S DNA solution, C20S DNA solution, C21S DNA solution, C22SDNA solution and C23S DNA solution, respectively. Five micrograms ofexpression vector pCEP 4 (Invitrogen) were digested with restrictionenzymes BamHI and XhoI. A DNA fragment with an approximate size of 10 kbwas purified and dissolved in 40 μL of sterile distilled water. This DNAsolution was designated as pCEP 4 DNA solution. One microliter of pCEP 4DNA solution and 6 μL of either C19S DNA solution, C20S DNA solution,C21S DNA solution, C22S DNA solution or C23S DNA solution wereindependently mixed with 7 μL of ligation buffer I from a DNA ligationkit ver. 2 and the ligation reactions were carried out. Competent E.coli strain DH5 α cells (100 μL) were transformed with 7 μL of eachligation mixture. Ampicillin-resistant transformants were screened forclones containing plasmid in which a 1.6-kb cDNA fragment is insertedbetween the recognition sites of BamHI and XhoI of pCEP 4 by analyzingthe DNA structure. The plasmids which were obtained containing the cDNAencoding OCIF-C19S, OCIF-C20S, OCIF-C21S, OCIF-C22S and OCIF-C23S (SEQID NOs: 83, 84, 85, 86, and 87, respectively) were designatedpCEP4-OCIF-C19S, pCEP4-OCIF-C20S, pCEP4-OCIF-C21S, pCEP4-OCIF-C22S andpCEP4-OCIF-C23S, respectively.

iii) Preparation of Domain-deletion Mutants of OCIF

(1) Deletion Mutagenesis of OCIF cDNA

A series of OCIF mutants with deletions from Thr 2 to Ala 42, from Pro43 to Cys 84, from Glu 85 to Lys 122, from Arg 123 to Cys 164, from Asp177 to Gln 251 or from Ile 252 to His 326 were prepared (positions ofthe amino acid residues are shown in SEQ ID NO: 4). These mutants weredesignated as OCIF-DCR1, OCIF-DCR2, OCIF-DCR3, OCIF-DCR4, OCIF-DDD1 andOCIF-DDD2, respectively, and assigned SEQ ID NOs: 67, 68, 69, 70, 71,and 72, respectively.

Mutagenesis was performed by two-step PCR as described in EXAMPLE22-ii). The primer sets for the reactions are shown in Table 11 and thenucleotide sequences of the primers are shown in SEQ ID NOs: 19, 25,40-53 and 54.

TABLE 11 mutants primer-1 primer-2 primer-3 primer-4 OCIF-DCR1 XhoI FDCR1R IF 2 DCR1F OCIF-DCR2 XhoI F DCR2R IF 2 DCR2F OCIF-DCR3 XhoI FDCR3R IF 2 DCR3F OCIF-DCR4 XhoI F DCR4R IF 16 DCR4F OCIF-DDD1 IF 8 DDD1RIF 14 DDD1F OCIF-DDD2 IF 8 DDD2R IF 14 DDD2F

The final PCR products were precipitated with ethanol, dried undervacuum and dissolved in 40 μL of sterile distilled water. Solutions ofDNA fragments coding for portions of OCIF-DCR1, OCIF-DCR2, OCIF-DCR3,OCIF-DCR4, OCIF-DDD1 and OCIF-DDD2 were designated DNA solutions F, G,H, I, J and K, respectively.

The DNA fragment contained in solution F (20 μL) was digested withrestriction enzymes NdeI and XhoI. A DNA fragment with an approximatesize of 500 bp was extracted from a preparative agarose gel using aQIAEX™ gel extraction kit and dissolved in 20 μL of sterile distilledwater. This DNA solution was designated DNA solution 11. Two microgramsof pSK+-OCIF were digested with restriction enzymes NdeI and XhoI. A DNAfragment with an approximate size of 4.0 kb was extracted from apreparative agarose gel using a QIAEX™ gel extraction kit and dissolvedin 20 μL of sterile distilled water. This DNA solution was designatedDNA solution 12. Two microliters of DNA solution 11, 3 μL of DNAsolution 12 and 5 μL of ligation buffer I from a DNA ligation kit ver. 2were mixed and the ligation was carried out. Competent E. coli strainDH5 α cells were transformed with 5 μL of the ligation mixture.Ampicillin-resistant transformants were screened for a clone containingplasmid DNA. DNA structure was analyzed by restriction enzyme mappingand by DNA sequencing. The plasmid thus obtained was namedpSK-OCIF-DCR1.

The DNA fragment which is contained in solution G (20 μL) was digestedwith restriction enzymes NdeI and XhoI. A DNA fragment with anapproximate size of 500 bp was extracted from a preparative agarose gelusing a QIAEX™ gel extraction kit and dissolved in 20 μL of steriledistilled water. This DNA solution was designated DNA solution 13. Twomicroliters of DNA solution 13, 3 μL of DNA solution 12 and 5 μL ofligation buffer I from a DNA ligation kit ver. 2 were mixed and ligationwas carried out. Competent E. coli strain DH5 α cells were transformedwith 5 μL of the ligation mixture. Ampicillin-resistant transformantswere screened for a clone containing a plasmid DNA. DNA structure wasanalyzed by restriction enzyme mapping and by DNA sequencing. Theplasmid thus obtained was named pSK-OCIF-DCR2.

The DNA fragment contained in solution H (20 μL) was digested withrestriction enzymes NdeI and XhoI. A DNA fragment with an approximatesize of 500 bp was extracted from a preparative agarose gel using aQIAEX™ gel extraction kit and dissolved in 20 μL of sterile distilledwater. This DNA solution was designated DNA solution 14. Two microlitersof DNA solution 14, 3 μL of DNA solution 12 and 5 μL of ligation bufferI from a DNA ligation kit ver. 2 were mixed and the ligation reactionwas carried out. Competent E. coli strain DH5 α cells were transformedwith 5 μL of the ligation mixture. Ampicillin-resistant transformantswere screened for a clone containing a plasmid DNA. DNA structure wasanalyzed by restriction enzyme mapping and by DNA sequencing. Theplasmid thus obtained was named pSK-OCIF-DCR3.

The DNA fragment contained in solution I (20 μL) was digested withrestriction enzymes XhoI and SphI. A DNA fragment with an approximatesize of 900 bp was extracted from a preparative agarose gel using aQIAEX™ gel extraction kit and dissolved in 20 μL of sterile distilledwater. This DNA solution was designated DNA solution 15. Two microgramsof pSK+-OCIF were digested with restriction enzymes XhoI and SphI. A DNAfragment with an approximate size of 3.6 kb was extracted from apreparative agarose gel using a QIAEX™ gel extraction kit and dissolvedin 20 μL of sterile distilled water. This DNA solution was designatedDNA solution 16. Two microliters of DNA solution 15, 3 μL of DNAsolution 16 and 5 μL of ligation buffer I from a DNA ligation kit ver. 2were mixed and the ligation reaction was carried out. Competent E. colistrain DH5 α cells were transformed with 5 μL of the ligation mixture.Ampicillin-resistant transformants were screened for a clone containingplasmid DNA. DNA structure was analyzed by restriction enzyme mappingand by DNA sequencing. The plasmid thus obtained was namedpSK-OCIF-DCR4.

The DNA fragment contained in solution J (20 μL) was digested withrestriction enzymes BstPI and NdeI. A DNA fragment with an approximatesize of 400 bp was extracted from a preparative agarose gel using aQIAEX™ gel extraction kit and dissolved in 20 μL of sterile distilledwater. This DNA solution was designated DNA solution 17. Two microlitersof DNA solution 17, 3 μL of DNA solution 8 and 5 μL of ligation buffer Ifrom a DNA ligation kit ver. 2 were mixed and the ligation reaction wascarried out. Competent E. coli strain DH5 α cells were transformed with5 μL of the ligation mixture. Ampicillin-resistant transformants werescreened for a clone containing plasmid DNA. DNA structure was analyzedby restriction enzyme mapping and by DNA sequencing. The plasmid thusobtained was named pSK-OCIF-DDD1.

The DNA fragment contained in solution K (20 μL) was digested withrestriction enzymes NdeI and BstPI. A DNA fragment with an approximatesize of 400 bp was extracted from a preparative agarose gel using aQIAEX™ gel extraction kit and dissolved in 20 μL of sterile distilledwater. This DNA solution was designated DNA solution 18. Two microlitersof DNA solution 18, 3 μL of DNA solution 8 and 5 μL of ligation buffer Ifrom a DNA ligation kit ver. 2 were mixed and the ligation reaction wascarried out. Competent E. coli strain DH5 α cells were transformed with5 μL of the ligation mixture. Ampicillin-resistant transformants werescreened for a clone containing plasmid DNA. DNA structure was analyzedby restriction enzyme mapping and by DNA sequencing. The plasmid thusobtained was named pSK-OCIF-DDD2.

2) Construction of Vectors for Expressing the OCIF Mutants

pSK-OCIF-DCR1, pSK-OCIF-DCR2, pSK-OCIF-DCR3, pSK-OCIF-DCR4,pSK-OCIF-DDD1 and pSK-OCIF-DDD2 were digested with restriction enzymesBamHI and XhoI. The BamHI-XhoI DNA fragment containing the entire codingsequence for each OCIF mutant was isolated and dissolved in 20 μL ofsterile distilled water. These DNA solutions that contain the BamHI-XhoIfragment derived from pSK-OCIF-DCR1, pSK-OCIF-DCR2, pSK-OCIF-DCR3,pSK-OCIF-DCR4, pSK-OCIF-DDD1 and pSK-OCIF-DDD2 were designated DCR1 DNAsolution, DCR2 DNA solution, DCR3 DNA solution, DCR4 DNA solution, DDD1DNA solution and DDD2 DNA solution, respectively. One microliter of pCEP4 DNA solution and 6 μL of either DCR1 DNA solution, DCR2 DNA solution,DCR3 DNA solution, DCR4 DNA solution, DDD1 DNA solution or DDD2 DNAsolution were independently mixed with 7 μL of ligation buffer I from aDNA ligation kit ver. 2 and the ligation reactions were carried out.Competent E. coli strain DH5 α cells (100 μL) were transformed with 7 μLof each ligation mixture. Ampicillin-resistant transformants werescreened for a clone containing plasmid DNA in which the DNA fragmentwith deletions is inserted between the recognition sites of BamHI andXhoI of pCEP 4 by analyzing the DNA structure. The plasmids containingthe cDNA encoding OCIF-DCR1, OCIF-DCR2, OCIF-DCR3, OCIF-DCR4, OCIF-DDD1and OCIF-DDD2 (SEQ ID NOs: 88, 89, 90, 91, 92, and 93, respectively)were designated pCEP4-OCIF-DCR1, pCEP4-OCIF-DCR2, pCEP4-OCIF-DCR3,pCEP4-OCIF-DCR4, pCEP4-OCIF-DDD1 and pCEP4-OCIF-DDD2, respectively.

iv) Preparation of OCIF with C-terminal Domain Truncation

(1) Mutagenesis of OCIF cDNA

A series of OCIF mutants with deletions from Cys at amino acid residue379 to Leu 380, from Ser 331 to Leu 380, from Asp 252 to Leu 380, fromAsp 177 to Leu 380, from Arg 123 to Leu 380 and from Cys 86 to Leu 380was prepared. Positions of the amino acid residues are shown in SEQ IDNO: 4. These mutants were designated as OCIF-CL, OCIF-CC, OCIF-CDD2,OCIF-CDD1, OCIF-CCR4 and OCIF-CCR3, respectively, and assigned SEQ IDNOs: 73, 74, 75, 76, 77, and 78, respectively.

Mutagenesis for OCIF-CL was performed by the two-step PCR as describedin EXAMPLE 22-ii). The primer set for the reaction is shown in Table 12.The nucleotide sequences of the primers are shown in SEQ ID NOs: 23, 40,55, and 66. The final PCR products were precipitated with ethanol, driedunder vacuum and dissolved in 40 μL of sterile distilled water. This DNAsolution was designated solution L.

The DNA fragment contained in solution L (20 μL) was digested withrestriction enzymes BstPI and EcoRV. A DNA fragment with an approximatesize of 100 bp was extracted from a preparative agarose gel using aQIAEX™ gel extraction kit and dissolved in 20 μL of sterile distilledwater. This DNA solution was designated DNA solution 19. Two microlitersof DNA solution 19, 3 μL of DNA solution 10 (described in EXAMPLE22-ii)) and 5 μL of ligation buffer I from a DNA ligation kit ver. 2were mixed and ligation reaction was carried out. Competent E. colistrain DH5 α cells were transformed with 5 μL of the ligation mixture.Ampicillin-resistant transformants were screened for a clone containingplasmid DNA. DNA structure was analyzed by restriction enzyme mappingand by DNA sequencing. The plasmid thus obtained was named pSK-OCIF-CL.Mutagenesis of OCIF cDNA to prepare OCIF-CC, OCIF-CDD2, OCIF-CDD1,OCIF-CCR4 and OCIF-CCR3 was performed by a one-step PCR reaction.

PCR reactions for mutagenesis to prepare OCIF-CC, OCIF-CDD2, OCIF-CDD1,OCIF-CCR4 and OCIF-CCR3 were as follows:

10× Ex Taq Buffer (Takara Shuzo) 10 μl 2.5 mM solution of dNTPs 8 μl theplasmid vector containing the entire OCIF cDNA 2 μl described in EXAMPLE11 (8 ng/ml) sterile distilled water 73.5 μl 20 μM solution of primerOCIF Xho F 5 μl 100 μM solution of primer (for mutagenesis) 1 μl Ex Taq(Takara Shuzo) 0.5 μl

TABLE 12 mutants primer-1 primer-2 primer-3 primer-4 OCIF-CL IF 6 CL RIF 14 CL F

Specific primers were used for each mutagenesis and other componentswere unchanged.

Primers used for the mutagenesis are shown in Table 13. Their nucleotidesequences are shown in SEQ ID NOs: 57-61. The components of each PCRwere mixed in a microcentrifuge tube and PCR was performed as follows.The microcentrifuge tubes were treated for 3 minutes at 97° C. and thenincubated sequentially, for 30 seconds at 95° C., 30 seconds at 50° C.and 3 minutes at 70° C. This three-step incubation procedure wasrepeated 25 times, and after that, the tubes were incubated for 5minutes at 70° C. An aliquot of the reaction mixture was removed fromeach tube and analyzed by agarose gel electrophoresis to confirm thesize of each product.

Excess primers in the PCRs were removed using an Amicon MICROCON™(Amicon) after completion of the reaction. The DNA fragments wereprecipitated with ethanol, dried under vacuum and dissolved in 40 μL ofsterile distilled water. The DNA fragment in each DNA solution wasdigested with restriction enzymes XhoI and BamHI. After the reactions,DNA was precipitated with ethanol, dried under vacuum and dissolved in20 μL of sterile distilled water.

The solutions containing the DNA fragment with the CC deletion, the CDD2deletion, the CDD1 deletion, the CCR4 deletion and the CCR3 deletionwere designated CC DNA solution, CDD2 DNA solution, CDD1 DNA solution,CCR4 DNA solution and CCR3 DNA solution, respectively.

TABLE 13 mutants primers for the mutagenesis OCIF-CC CC R OCIF-CDD2 CDD2R OCIF-CDD1 CDD1 R OCIF-CCR4 CCR4 R OCIF-CCR3 CCR3 R

(2) Construction of Vectors for Expressing the OCIF Mutants

pSK-OCIF-CL was digested with restriction enzymes BamHI and XhoI. TheBamHI-XhoI DNA fragment containing the entire coding sequence forOCIF-CL was isolated and dissolved in 20 μL of sterile distilled water.This DNA solution was designated CL DNA solution. One microliter of pCEP4 DNA solution and 6 μL of either CL DNA solution, CC DNA solution, CDD2DNA solution, CDD1 DNA solution, CCR4 DNA solution or CCR3 DNA solutionwere independently mixed with 7 μL of ligation buffer I from a DNAligation kit ver. 2 and the ligation reactions were carried out.Competent E. coli strain DH5α cells (100 μL) were transformed with 7 μLof each ligation mixture. Ampicillin-resistant transformants werescreened for clones containing plasmids which have the desirablemutations in the OCIF cDNA by analyzing the DNA structure. In eachplasmid, the OCIF cDNA fragment having a deletion was inserted betweenthe recognition sites of XhoI and BamHI of pCEP 4. The plasmidscontaining the cDNA encoding OCIF-CL, OCIF-CC, OCIF-CDD1, OCIF-CDD2,OCIF-CCR4 and OCIF-CCR3 (SEQ ID NOs: 94, 95, 96, 97, 98, and 99,respectively) were designated pCEP4-OCIF-CL, pCEP4-OCIF-CC,pCEP4-OCIF-CDD2, pCEP4-OCIF-CDD1, pCEP4-OCIF-CCR4 and pCEP4-OCIF-CCR3,respectively.

v) Preparation of OCIF Mutants with C-terminal Truncations

(1) Introduction of C-terminal Truncations to OCIF

A series of OCIF mutants with C-terminal truncations was prepared. AnOCIF mutant in which 10 residues from Gln at 371 to Leu at 380 werereplaced with 2 residues (Leu-Val) was designated OCIF-CBst (SEQ ID NO:79). An OCIF mutant in which 83 residues from Cys 298 to Leu 380 werereplaced with 3 residues (Ser-Leu-Asp) was designated OCIF-CSph (SEQ IDNO: 80). An OCIF mutant in which 214 residues from Asn 167 to Leu 380were removed was designated OCIF-CBsp (SEQ ID NO: 81). An OCIF mutant inwhich 319 residues from Asp 62 to Leu 380 were replaced with 2 residues(Leu-Val) was designated OCIF-CPst (SEQ ID NO: 82). Positions of theamino acid residues are shown in SEQ ID NO: 4.

Two micrograms each of pSK+-OCIF were digested with BstPI, SphI, PstI(Takara Shuzo) or BspEI (New England Biolabs) followed by phenolextraction and ethanol precipitation. The precipitated DNA was dissolvedin 10 μL of sterile distilled water. The ends of the DNAs in 2 μL ofeach solution were blunted using a DNA blunting kit in a final volume of5 μL. To the reaction mixtures, 1 μg (1 μL) of an Amber codon-containingXbaI linker (5′-CTAGTCTAGACTAG-3′) and 6 μL of ligation buffer I from aDNA ligation kit ver. 2 were added.

After the ligation reactions, 6 μL each of the reaction mixtures wasused to transform E. coli strain DH5 α. Ampicillin-resistanttransformants were screened for clones containing plasmids. DNAstructure was analyzed by restriction enzyme mapping and by DNAsequencing. The plasmids thus obtained were named pSK-OCIF-CBst,pSK-OCIF-CSph, pSK-OCIF-CBsp and pSK-OCIF-CPst, respectively.

(2) Construction of Vectors Expressing the OCIF Mutants

pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp and pSK-OCIF-CPst weredigested with restriction enzymes BamHI and XhoI. The 1.5 kb DNAfragment containing the entire coding sequence for each OCIF mutant wasisolated and dissolved in 20 μL of sterile distilled water. These DNAsolutions that contained the BamHI-XhoI fragment derived frompSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp or pSK-OCIF-CPst weredesignated CBst DNA solution, CSph DNA solution, CBsp DNA solution andCPst DNA solution, respectively. One microliter of pCEP 4 DNA solution(described in EXAMPLE 22-ii)) and 6 μL of either CBst DNA solution, CSphDNA solution, CBsp DNA solution or CPst DNA solution were independentlymixed with 7 μL of ligation buffer I from a DNA ligation kit ver. 2 andthe ligation reactions were carried out. Competent E. coli strain DH5 αcells (100 μL) were transformed with 7 μL of each ligation mixture.Ampicillin-resistant transformants were screened for clones containingplasmids in which the cDNA fragment was inserted between the recognitionsites of BamHI and XhoI of pCEP 4 by analyzing the DNA structure. Theplasmids containing the cDNA encoding OCIF-CBst, OCIF-CSph, OCIF-CBsp orOCIF-CPst (SEQ ID NOs: 100, 101, 102, and 103, respectively) weredesignated pCEP4-OCIF-CBst, pCEP4-OCIF-CSph, pCEP4-OCIF-CBsp andpCEP4-OCIF-CPst, respectively.

vi) Preparation of Vectors for Expressing the OCIF Mutants

E. Coli clones harboring the expression vectors for OCIF mutants (atotal of 21 clones) were grown and the vectors were purified by usingQIAGEN™ columns (QIAGEN). All the expression vectors were precipitatedwith ethanol and dissolved in appropriate volumes of sterile distilledwater and used for further manipulations shown below.

vii) Transient Expression of the cDNAs for OCIF Mutants and BiologicalActivities of the Mutants

OCIF mutants were produced using the expression vectors prepared inEXAMPLE 22-vi). The method was essentially the same as described inEXAMPLE 13. Only the modified points are described below. 2×10⁵ cells of293/EBNA suspended in IMDM containing 10% fetal bovine serum were seededinto each well of a 24 well plate. One microgram of purified vector DNAand 4 μL of lipofectamine were used for each transfection. A mixture ofthe expression vector and lipofectamine in OPTI-MEM™ (GIBCO BRL) in afinal volume of 0.5 ml was added to the cells in a well. After the cellswere incubated at 37° C. for 24 hr in 5% CO₂, the medium was replacedwith 0.5 ml of EXCELL™ 301 medium (JSR). The cells were incubated at 37°C. for a further 48 hours in 5% CO₂. The conditioned medium wascollected and used in assays for in vitro biological activity. Thenucleotide sequences of cDNAs for the OCIF mutants are shown in SEQ IDNOs: 83-103. The deduced amino acid sequences for the OCIF mutants areshown in SEQ ID NOs: 62-82. The assay for in vitro biological activitywas performed as described in EXAMPLE 13. The antigen concentration ofeach conditioned medium was determined by ELISA as described in EXAMPLE24. Table 14 shows the activity of each mutant relative to that of theunaltered OCIF.

TABLE 14 mutants activity the unaltered OCIF ++ OCIF-C19S + OCIF-C20S ±OCIF-C21S ± OCIF-C22S + OCIF-C23S ++ OCIF-DCR1 ± OCIF-DCR2 ± OCIF-DCR3 ±OCIF-DCR4 ± OCIF-DDD1 + OCIF-DDD2 ± OCIF-CL ++ OCIF-CC ++ OCIF-CDD2 ++OCIF-CDD1 + OCIF-CCR4 ± OCIF-CCR3 ± OCIF-CBst ++ OCIF-CSph ++ OCIF-CBsp± OCIF-CPst ± ++ indicates relative activity more than 50% of that ofthe unaltered OCIF; + indicates relative activity between 10% and 50%; ±indicates relative activity less than 10%, or production level too lowto determine the accurate biological activity.

viii) Western Blot Analysis

Ten microliters of the final conditioned medium was used for westernblot analysis. Ten microliters of each sample were mixed with 10 μL ofSDS-PAGE sample buffer (0.5 M Tris-HCl, 20% glycerol, 4% SDS, 20 μg/mlbromophenol blue, pH 6.8), boiled for 3 min. and subjected to 10% SDSpolyacrylamide gel electrophoresis under non-reducing conditions. Afterthe electrophoresis, the separated proteins were blotted to PVDFmembrane (PROBLOTT™, Perkin Elmer) using a semi-dry electroblotter(BIO-RAD). The membrane was incubated at 37° C. with horseradishperoxidase-labeled anti-OCIF antibodies for 2 hr. After the membrane waswashed, protein bands which react with the labeled antibodies weredetected using an ECL™ system (Amersham). Two protein bands withapproximate molecular weights of 60 kD and 120 kD were detected for theunaltered OCIF. On the other hand, almost exclusively a 60 kD proteinband was detected for the OCIF-C23S, OCIF-CL and OCIF-CC mutants.Protein bands with approximate weights of 40 kD-50 kD and 30 kD-40 kDwere the major ones for OCIF-CDD2 and OCIF-CDD1, respectively. Theseresults indicate that Cys at 379 is responsible for the dimer formation,both the monomers and the dimers maintain the biological activity and adeletion of residues from Asp at 177 to Leu at 380 does not abolish thebiological activity of OCIF (positions of the amino acid residues areshown in SEQ ID NO: 4).

EXAMPLE 23

Isolation of Human Genomic OCIF Gene

i) Screening of a Human Genomic Library

An amplified human placenta genomic library in LAMBDA FIX™ II vector(Stratagene) was screened for the gene encoding human OCIF using thehuman OCIF cDNA as a probe. Essentially, screening was done according tothe instruction manual supplied with the genomic library. The basicprotocols described in Molecular Cloning: A Laboratory Manual were alsoemployed to manipulate phage, E. coli, and DNA.

The library was titered, and 1×10⁶ pfu of phage was mixed with XL1-BlueMRA host E. coli cells and plated onto 20 plates (9 cm×13 cm) with 9 mlper plate of top agarose. The plates were incubated overnight at 37° C.Filter plaque lifts were prepared using HYBOND™-N nylon membranes(Amersham). The membranes were processed by denaturation in a solutioncontaining 1.5 M NaCl and 0.5 M NaOH for 1 minute at room temperature.The membranes were then neutralized by placing each one in 1 M Tris-HCl(pH 7.5) and a solution containing 1.5 M NaCl and 0.5 M Tris-HCl (pH7.5) successively for one minute each. The membranes were thentransferred onto a filter paper wetted with 2×SSC. Phage DNA was fixedonto the membranes with 1200 microJoules of UV energy using aSTRATALINKER UV CROSSLINKER™ 2400 (STRATAGENE) and the membranes wereair dried. The membranes were immersed in Rapid Hybridization buffer(Amersham) and incubated for one hour at 65° C. before hybridizationwith ³²P-labeled cDNA probe in the same buffer overnight at 65° C.Screening probe was prepared by labeling the OCIF cDNA with ³²P usingthe Megaprime DNA labeling system (Amersham). Approximately, 5×10⁵ cpmprobe was used for each ml of hybridization buffer. After thehybridization, the membranes were rinsed in 2×SSC for five minutes atroom temperature. The membranes were then washed four times, 20 minuteseach time, in 0.5×SSC containing 0.1% SDS at 65° C. After the finalwash, the membranes were dried and subjected to autoradiography at −80°C. with SUPER HR-H™ X-ray film (FUJI PHOTO FILM Co., Ltd.) and anintensifying screen.

Upon examination of the autoradiograms, six positive signals weredetected. Agar plugs were picked from the regions corresponding to thesesignals for phage purification. Each agar plug was soaked overnight in0.5 ml of SM buffer containing 1% chloroform to extract phage. Eachextract containing phage was diluted 1000 fold with SM buffer and analiquot of 1 μL or 20 μL was mixed with host E. coli described above.The mixture was plated onto agar plates with top agarose as describedabove. The plates were incubated overnight at 37° C., and filter liftswere prepared, prehybridized, hybridized, washed and autoradiographed asdescribed above. This process of phage purification was applied to allsix positive signals initially detected on the autoradiograms and wasrepeated until all phage plaques on agar plates hybridize with the cDNAprobe. After purification, agar plugs of each phage isolate were soakedin SM buffer containing 1% chloroform and stored at 4° C. Six individualphage isolates were designated λ0IF3, λ0IF8, λ0IF9, λ0IF11, λ0IF12 andλ0IF17, respectively.

ii) Analysis of the Genomic Clones by Restriction Enzyme Digestion andSouthern Blot Hybridization

DNA was prepared from each phage isolate by the plate lysate method asdescribed in Molecular Cloning: A Laboratory Manual. DNA prepared fromeach phage was digested with restriction enzymes and the fragmentsderived from the digestion were separated on agarose gels. The fragmentswere then transferred to nylon membranes and subjected to Southern blothybridization using OCIF cDNA as a probe. The results of the analysisrevealed that the six phage isolates are individual clones. Among thesefragments derived from restriction enzyme digestion, those fragmentswhich hybridized with the OCIF cDNA probe were subcloned into plasmidvectors and subjected to nucleotide sequence analysis as describedbelow.

iii) Subcloning Restriction Fragments Derived from Genomic Clones intoPlasmid Vectors and Determining Their Nucleotide Sequence.

λ0IF8 DNA was digested with restriction enzymes EcoRI and NotI and theDNA fragments derived therefrom were separated on a 0.7% agarose gel.The 5.8 kilobase pair (kb) EcoRI/NotI fragment was extracted from thegel using a QIAEX™ II Gel Extraction Kit (QIAGEN) according to theprocedure recommended by the manufacturer. The 5.8 kb EcoRI/NotIfragment was ligated with pBLUESCRIPT II SK+™ vector (STRATAGENE), whichhad been linearized with restriction enzymes EcoRI and NotI, usingREADY-TO-GO™ T4 DNA Ligase (Pharmacia) according to the procedurerecommended by the manufacturer. Competent DH5 α E. coli cells(Amersham) were transformed with the recombinant plasmid andtransformants were selected on L-plates containing 50 μg/ml ofampicillin.

A clone harboring the recombinant plasmid containing the 5.8 kbEcoRI/NotI fragment was isolated and this plasmid was termed pBSG8-5.8.pBSG8-5.8 was digested with HindIII and a 0.9 kb DNA fragment derivedfrom this digestion was isolated in the same manner as described above.This 0.9 kb fragment was then cloned into pBLUESCRIPT II SK−™ at theHindIII site as described above. This recombinant plasmid containing 0.9kb HindIII fragment was denoted pBS8H0.9.

λ0IF11 DNA was digested with EcoRI and 6 kb, 3.6 kb, 2.6 kb EcoRIfragments were isolated in the same manner as described above and clonedinto a pBLUESCRIPT II SK+™ vector at the EcoRI site as described above.These recombinant plasmids were termed pBSG11-6, pBSG11-3.6, andpBSG11-2.6, respectively. pBSG11-6 was digested with HindIII and thedigest was separated on a 0.7% agarose gel. Three fragments, 2.2 kb, 1.1kb, and 1.05 kb in length, were extracted from the gel and clonedindependently into pBLUESCRIPT II SK−™ vector at the HindIII site in thesame manner as described above. These recombinant plasmids were termedpBS6H2.2, pBS6H1.1 and pBS6H1.05, respectively.

The nucleotide sequence of the cloned genomic DNA was determined using aABI DYEDEOXY TERMINATOR CYCLE SEQUENCING READY REACTION™ Kit (PERKINELMER) and a 373A DNA Sequencing system (Applied Biosystems). PlasmidspBSG8-5.8, pBS8H0.9, pBSG11-6, pBSG11-3.6, pBSG11-2.6, pBS6H2.2,pBS6H1.1 and pBS6H1.05 were prepared according to the alkaline-SDSprocedure as described in Molecular Cloning: A Laboratory Manual andused as templates for DNA sequence analysis. The nucleotide sequence ofthe human OCIF gene is presented in SEQ ID NO: 104 and SEQ ID NO: 105.The nucleotide sequence of the DNA, between exon 1 and exon 2, was notentirely determined. There is a stretch of approximately 17 kb betweenthe sequences given in SEQ ID NO: 104 and SEQ ID NO: 105.

EXAMPLE 24

Quantitation of OCIF by EIA

i) Preparation of Anti-OCIF Antibody

Male Japanese white rabbits (Kitayama Labs Co., LTD) weighing 2.5-3.0 kgwere used in immunization for preparing antisera. For immunization, anemulsion was prepared by mixing an equal volume of rOCIF (200 μg/ml) andcomplete Freund's adjuvant (Difco, Cat. 0638-60-7). Three rabbits wereimmunized subcutaneously six times at one week intervals with 1 ml ofemulsion per injection. Whole blood was obtained ten days after thefinal immunization and serum was isolated. Antibody was purified fromserum as follows. Antiserum was diluted two-fold with PBS. After addingammonium sulfate at a final concentration of 40% w/v, the antiserum wasallowed to stand at 4° C. for 1 hr. The precipitate obtained bycentrifugation at 8000×g for 20 min. was dissolved in a small volume ofPBS and was dialyzed against PBS. The resultant solution was loaded ontoa Protein G-SEPHAROSE™ column (Pharmacia). After washing with PBS,absorbed immunoglobulin G was eluted with 0.1 M glycine-HCl buffer (pH3.0). The eluate was immediately neutralized with 1.5 M Tris-HCl buffer(pH 8.7) and dialyzed against PBS. Protein concentration was determinedby absorbance at 280 nm (E^(1%) 13.5).

Horseradish peroxidase-labeled antibody was prepared using anIMMUNOPURE™ Maleimide Activated Horseradish Peroxidase Kit (Pierce, Cat.31494). Briefly, one mg of IgG was incubated with 80 μg ofN-succinimidyl-S-acetylthioacetate for 30 min. After deacetylation with5 mg of hydroxylamine HCl, modified IgG was separated using apolyacrylamide desalting column. The protein pool was mixed with one mgof maleimide-activated horseradish peroxidase and incubated at roomtemperature for 1 hr.

ii) Quantitation of OCIF by Sandwich EIA

Microtiter plates (Nunc MaxiSorp Immunoplate) were coated with rabbitanti-OCIF IgG by incubating 0.2 μg in 100 μL of 50 mM sodium bicarbonatebuffer pH 9.6 at 4° C. overnight. After blocking the plates byincubating for 1 hour at 37° C. with 300 μL of 25% BLOCKACE™/PBS (SnowBrand Milk Products), 100 μL samples were incubated for 2 hours at roomtemperature. After washing the plates three times with PBST (PBScontaining 0.05% TWEEN™ 20), 100 μL of 1:10000 diluted horseradishperoxidase-labeled anti-OCIF IgG was added and incubated for 2 hours atroom temperature. The amount of OCIF was determined by incubation with100 μL of a substrate solution (TMB, ScyTek Lab., Cat. TM4999) andmeasurement of the absorbance at 450 nm using an IMMUNOREADER™ (NuncNJ2000). Purified recombinant OCIF was used as a standard protein and atypical standard curve is shown in FIG. 13.

EXAMPLE 25

Anti-OCIF Monoclonal Antibody

i) Preparation of a Hybridoma Producing Anti-OCIF Monoclonal Antibody.

OCIF was purified to homogeneity from the culture medium of humanfibroblasts, IMR-90 cells by the purification method described inEXAMPLE 11. Purified OCIF was dissolved in PBS at a concentration of 10μg/100 μL. BALB/c mice were immunized by administering this solutionintraperitoneally three times every two weeks. In the first and thesecond immunizations, the emulsion was composed of an equal volume ofOCIF and Freund's complete adjuvant. Three days after the finalimmunization, the spleen was removed and lymphocytes isolated and fusedwith mouse myeloma p3x63-Ag8.653 cells according to conventional methodsusing polyethyleneglycol. Then the fused cells were cultured in HATmedium to select hybridomas. The presence of anti-OCIF antibody in theculture medium of each hybridoma was determined by solid phase ELISA.Briefly, each well of a 96-well immunoplate (Nunc) was coated with 100μL of purified OCIF (10 μg/ml in 0.1 M NaHCO₃) and blocked with 50%BLOCKACE™ (Snow Brand Milk Products Co. Ltd.). The hybridoma clonessecreting anti-OCIF antibody were established by limit dilution cloning3-5 times and by solid phase ELISA screening. Several hybridoma clonesproducing high levels of anti-OCIF antibody were selected.

ii) Production of Anti-OCIF Monoclonal Antibodies.

Each hybridoma clone secreting anti-OCIF antibody obtained in EXAMPLE25-i) was transplanted intraperitoneally into mice given Pristane(Aldrich) at a cell density of 1×10⁶ cells/mouse. The accumulatedascites was collected 10-14 days after transplantation, therebyobtaining anti-OCIF specific monoclonal antibody of the presentinvention. Purified antibodies were obtained by Affigel protein ASEPHAROSE™ chromatography (BioRad) according to the manufacturer'smanual. Briefly, the ascites fluid was diluted with an equal volume of abinding buffer (BioRad) and applied to a protein A column. The columnwas washed with a sufficient volume of binding buffer and eluted with anelution buffer (BioRad). After neutralizing, the eluate obtained wasdialyzed in water and subsequently lyophilized. The purity of theantibody thereby obtained was analyzed by SDS/PAGE and a homogenous bandwith a molecular weight of about 150,000 was detected.

iii) Selection of Monoclonal Antibodies Having High Affinity for OCIF

Each antibody obtained in EXAMPLE 25-ii) was dissolved in PBS and theprotein concentration was determined by the method of Lowry. Eachantibody solution was diluted to the same concentration and thenserially diluted with PBS. Monoclonal antibodies, which can recognizeOCIF even at highly dilute concentrations, were selected by solid phaseELISA described in EXAMPLE 25-ii). Thus, three monoclonal antibodiesA1G5, E3H8 and D2F4 were selected.

iv) Determination of Class and Subclass of Antibodies

The class and subclass of the antibodies of the present inventionobtained in EXAMPLE 25-iii) were analyzed using an immunoglobulin classand subclass analysis kit (Amersham). The procedure was carried outaccording to the kit directions. The results are shown in Table 15. Theantibodies of the present invention, E3H8, A1G5 and D2F4 belong to theIgG₁, IgG_(2a) and IgG_(2b) subclasses, respectively.

TABLE 15 Analysis of class and subclass of the antibodies in the presentinvention. Antibody IgG₁ IgG_(2a) IgG_(2b) IgG₃ IgA IgM κ A1G5 − + − − −− + E3H8 + − − − − − + D2F4 − − + − − − +

v) Quantitation of OCIF by ELISA

Three kinds of monoclonal antibodies, A1G5, E3H8 and D2F4 obtained inEXAMPLE 25-iv), were used as solid phase antibodies and enzyme-labeledantibodies, respectively. Sandwich ELISA was constructed by differentcombinations of solid phase antibody and labeled antibody. The labeledantibody was prepared using an IMMUNOPURE™ TM Maleimide-ActivatedHorseradish Peroxidase Kit (Pierce, Cat. No. 31494). Each monoclonalantibody was dissolved in 0.1 M NaHCO₃ at a concentration of 10 μg/ml,and 100 μL of the solution was added to each well of a 96-wellimmunoplate (Nunc, MaxiSorp Cat. No. 442404) followed by allowing themto stand at room temperature overnight. Subsequently, each well of theplate was blocked with 50% BLOCKACE™ (Snow Brand Milk Products, Co.,Ltd.) at room temperature for 50 minutes, and washed three times withPBS containing 0.1% TWEEN™ 20 (washing buffer).

A series of concentrations of OCIF was prepared by diluting OCIF with1st reaction buffer (0.2 M Tris-HCl buffer, pH 7.4, containing 40%BLOCKACE™ and 0.1% TWEEN™ 20). Each well of a 96-well immunoplate wasfilled with 100 μL of the prepared OCIF solution with eachconcentration, allowed to stand at 37° C. for 3 hours, and subsequentlywashed three times with washing buffer. The POD-labeled antibody wasdiluted 400-fold with 2nd reaction buffer (0.1 M Tris-HCl buffer, pH7.4, containing 25% BLOCKACE™ and 0.1% TWEEN™ 20), and 100 μL of thediluted solution was added to each well of the immunoplates. Eachimmunoplate was allowed to stand at 37° C. for 2 hours, and subsequentlywashed three times with washing buffer. After washing, 100 μL of asubstrate solution (0.1 M citrate-phosphate buffer, pH 4.5, containing0.4 mg/ml of o-phenylenediamine HCl and 0.006% H₂O₂) was added to eachwell of the immunoplates and the immunoplates incubated at 37° C. for 15min. The enzyme reaction was terminated by adding 50 μL of 6 N H₂SO₄ toeach well. The optical density of each well was determined at 492 nmusing an immunoreader (IMMUNOREADER™ NJ 2000, Nunc).

Using three different monoclonal antibodies of the present invention,each combination of solid phase and POD-labeled antibodies leads to anaccurate determination of OCIF concentration. Each monoclonal antibodyof the present invention was confirmed to recognize a different epitopeof OCIF. A typical standard curve of OCIF using a combination of solidphase antibody, A1G5, and POD-labeled antibody, E3H8, is shown in FIG.14.

vi) Determination of OCIF in Human Serum

The concentration of OCIF in five samples of normal human serum wasdetermined using an EIA system described in EXAMPLE 25-v). Theimmunoplates were coated with A1G5 as described in EXAMPLE 25-v), and 50μL of the 1^(st) reaction buffer was added to each well of theimmunoplates. Subsequently, 50 μL of each human serum was added to eachwell of the immunoplates. The immunoplates were incubated at 37° C. for3 hours and washed three times with washing buffer. After washing, eachwell of the immunoplates was filled with 100 μL of POD-E3H8 antibodydiluted 400-fold with the 2^(nd) reaction buffer and incubated at 37° C.for 2 hours. After washing the immunoplates three times with washingbuffer, 100 μL of the substrate solution described in EXAMPLE 25-v) wasadded to each well and incubated at 37° C. for 15 min. The enzymereaction was terminated by adding 50 μL of 6 N H₂SO₄ to each well of theimmunoplates. The optical density of each well was determined at 492 nmusing an immunoreader (IMMUNOREADER™ NJ 2000, Nunc).

1^(st) reaction buffer containing the known amount of OCIF was treatedin the same way and a standard curve of OCIF as shown in FIG. 2 wasobtained. Using the standard curve of OCIF, the amount of OCIF in humanserum sample was determined. The results were shown in Table 16.

TABLE 16 The amount of OCIF in normal human serum Serum Sample OCIFConcentration (ng/ml) 1 5.0 2 2.0 3 1.0 4 3.0 5 1.5

EXAMPLE 26

Therapeutic Effect on Osteoporosis

(1) Method

Six week old male Fischer rats were subjected to denervation of the leftforelimb. These rats were assigned to four groups (10 rats/group) andtreated as follows: group A, sham operated rats without administration;group B, denerved rats with the vehicle administered intravenously;group C, denerved rats with OCIF administered intravenously at a dose of5 μg/kg twice a day; group D, denerved rats with OCIF administeredintravenously at a dose of 50 μg/kg twice a day. After denervation, OCIFwas administered daily for 14 days. After 2 weeks treatment, the animalswere sacrificed and their forelimbs were dissected. Thereafter boneswere tested for mechanical strength.

(2) Results

A decrease in bone strength was observed in control animals as comparedto those animals of the normal groups while bone strength was increasedin the group of animals that received 50 mg of OCIF per kg body weight.

Samples of the hybridomas that produce the claimed monoclonal antibodieswere deposited in the National Institute of Bioscience and HumanTechnology National Institute of Advanced Industrial Science andTechnology. The National Institute of Bioscience and Human TechnologyNational Institute of Advanced Industrial Science and Technologyaccession numbers for the deposited hybridomas are:

Hybridoma Antibody Designation Deposit Date Accession No. A1G5 Feb. 5,2001 FERM BP-7441 D2F4 Feb. 5, 2001 FERM BP-7442 E3H8 Feb. 5, 2001 FERMBP-7443

These deposits were made under the Budapest Treaty and will bemaintained and made accessible to others in accordance with theprovisions thereof.

The hybridomas will be maintained in a public depository for a term ofat least 30 years and at least five years after the most recent requestfor the furnishing of a sample of the deposit is received by thedepository. In any case, samples will be stored under agreements thatwould make them available beyond the enforceable life of any patentissuing from the above-referenced application.

INDUSTRIAL AVAILABILITY

The present invention provides both a novel protein which inhibits theformation of osteoclasts and an efficient procedure for producing theprotein. The protein of the present invention inhibits the formation ofosteoclasts. The protein will be useful for the treatment of manydiseases accompanied by bone loss, such as osteoporosis, and as anantigen to prepare antibodies useful for the immunological diagnosis ofsuch diseases.

A national deposit of the microorganism (accession number Bikkoken No.P-14998) was made on Jun. 21, 1995, and transferred to the internationaldepository named “National Institute of Bioscience and Human-Technology(NIBH), Agency of Industrial Science and Technology, Ministry ofInternational Trade and Industry” having an address of 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki-ken, 305, JAPAN on Oct. 25, 1995 asaccession number FERM BP-5267, pursuant to the Budapest Treaty. Thedeposit is a Budapest Treaty deposit and will be maintained and madeaccessible to others in accordance with the provisions thereof. Theinternational depository is currently known as “International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology”.

1. An isolated nucleic acid molecule comprising the sequence ofnucleotides 1 to 1206 of SEQ ID NO:6, inclusive, or the sequence ofnucleotides 64 to 1206 of SEQ ID NO:6.
 2. The nucleic acid molecule ofclaim 1 which is cDNA, genomic DNA or RNA.
 3. An isolated nucleic acidencoding a polypeptide comprising the amino acid sequence from residues1 to 380 of SEQ ID NO:4 or from residues 1 to 401 of SEQ ID NO:5.
 4. Thenucleic acid molecule of claim 3 which is cDNA, genomic DNA or RNA.
 5. AcDNA comprising the nucleotide sequence as provided in SEQ ID NO:6.
 6. AcDNA comprising the nucleotide sequence of nucleotides 64 to 1206 of SEQID NO:6.
 7. A cDNA encoding the amino acid sequence as provided in SEQID NO:4.
 8. A cDNA encoding the amino acid sequence as provided in SEQID NO:5.
 9. An expression vector comprising an isolated nucleic acidmolecule comprising the sequence of nucleotides 1 to 1206 of SEQ ID NO:6or the sequence of nucleotides 64 to 1206 of SEQ ID NO:6.
 10. Anexpression vector comprising an isolated nucleic acid molecule encodinga polypeptide comprising the amino acid sequence from residues 1 to 380of SEQ ID NO:4 or from residues 1 to 401 of SEQ ID NO:5.
 11. A host cellcomprising an expression vector comprising an isolated nucleic acidmolecule comprising the sequence of nucleotides 1 to 1206 of SEQ IDNO:6, inclusive, or the sequence of nucleotides 64 to 1206 of SEQ IDNO:6.
 12. A host cell comprising an expression vector comprising anisolated nucleic acid molecule encoding a polypeptide comprising theamino acid sequence from residues 1 to 380 of SEQ ID NO:4 or fromresidues 1 to 401 of SEQ ID NO:5.